crystals Precession photo NaCl


Introduction

The key step in a single crystal structure determination is to obtain the single crystal (!) and the better the crystal quality the more likely is a satisfactory outcome. A good crystal gives sharp diffracted beams but good visual indications are a well-formed crystal with sharp edges and flat faces, optically transparent and for most crystal systems a sharp extinction under the polarizing microscope [1]. In practice, frequently one has to work with less than ideal crystals.

No one can tell you how to obtain suitable crystals of your (new) material - indeed it may not be possible - but there are several well tried methods and there is plenty of scope for ingenuity. These notes are directed at small-molecule crystallography but biochemists, because of the difficulty in growing crystals of biomolecules, have been well served by reviews and monographs and the small-molecule chemist can profit from this expertise.

There are several important points to be aware of:

  • The crystals obtained may not have the composition of the starting sample. Perhaps the commonest example is the formation of a solvate where solvent molecules are incorporated into the crystal structure, but chemical reactions also occur.
  • If an impurity in your sample grows beautiful crystals, you may in error examine these (non-representative) crystals. Check that you are not selecting a crystal in a batch that is different from the bulk.
  • The usual advice is to work with a pure sample of material (particularly true for biomolecules) and to have solutions free from suspended matter. However see a provocative article by Hope [2] for another view.
  • Only a few crystals are required - the minimum is one (!) - and with a typical size 0.1–0.3 mm linear dimensions (mass circa 10-5 g) you can work on a small scale and perhaps try several different smaller experiments to increase the chance of success.
  • Good crystals form slowly over days, weeks or even months so you must be patient and not mechanically disturb the set up. Obviously you check to see what is happening.
  • It is much better to leave the crystals in the liquor that they have grown from [2]. This is sometimes difficult to do as they may (usually do) deteriorate on leaving in contact with liquid.
  • Record accurately what you do in case it is necessary to repeat the experiment. Note all solvents that have been in contact with the sample. I know you do this anyway but ...

Finally don't despair - growing crystals is a slow, frustrating business but persistence does pay off at least for most people some of the time.



Where to look

Several crystallographic textbooks have a section of a few pages dealing with crystal growth [3,4]. In addition there are a few short review articles aimed at the small-molecule chemist and covering a range of the more common methods. These include Jones [5] and van der Sluis et al. [6] with the latter directed particularly at the organic chemist. The study of crystal growth is a large and important field often directed at industrial processes and a wide-ranging review by Hulliger [7] provides examples and references to a variety of methods suitable for small-scale working.

People who hit on a novel method may publish a note in the Journal of Applied Crystallography and the Journal of Crystal Growth is also a useful source. For reviews on biochemical crystal growing see a later section.

There is an element of luck in finding descriptions of ingenious ideas to grow crystals where these are 'hidden' in the experimental part of a paper and of course do not appear in any abstract.

The two commonest methods currently used are the vapour-diffusion method and the liquid-diffusion (layering) method both of which are described in refs. 3 and 4 (and others). Typical experimental arrangements are described below but if you are part of a group already in the crystal growing game you should be able to see the techniques in use without picking up too many bad habits!

There are also web sites covering this topic and whilst these are largely covering the same ground each brings small points of technique or helpful asides based on the author's experiences. Web site URLs do tend to change but the following have been visited.

Dr Alexander J. Blake's page from the University of Nottingham. Also describes crystal mounting techniques.
     [http://www.nott.ac.uk/~pczajb2/growcrys.htm]
Paul D. Boyle's "Growing Crystals That Will Make Your Crystallographer Happy" (North Carolina State University).
     [http://www.xray.ncsu.edu/GrowXtal.html]
Martin Lutz's pages (University of Utrecht) - hints & tips, gels, bio, melts.
     [http://www.cryst.chem.uu.nl/growing.html]
Drs Dinger and Klosin (University of Florida) - brief list of hints and tips with examples.
     [http://xray.chem.ufl.edu/html/crystal_growing_tips.htm]



Vapour diffusion

In this experiment two miscible liquids are placed separately in a sealed container and transport of volatile solvents takes place between them via the vapour [3,4]. For example if the solid which you are trying to grow crystals of is soluble in CH2Cl2 but insoluble in (volatile) diethyl ether then the transport of diethyl ether into the CH2Cl2 solution would eventually produce solid (crystals hopefully) over days/weeks. In this case since CH2Cl2 is also volatile, transport of this species would also occur and only stop when both liquids have the same composition.

There are a large number of possible combinations with different solvent systems, solute concentration, temperature and the rate of transport by impeding the vapour path. Clearly some exploratory experiments to find suitable solvents may be required. Some typical experimental setups are shown in Fig. 1 - note that several tubes can be placed in one outer container. A very small-scale arrangement with the sample held in solution in a capillary has been described [10] but appears more complex than a sitting drop experiment.

  
Fig. 1(a) Fig. 1(b)
  


Liquid diffusion

In these experiment (sometimes called 'free-interface diffusion'), two miscible liquids are placed in contact such that there is a well-defined junction between them with little mixing. The solute to be crystallised (A) is dissolved in one of the liquids prior to setting up the experiment and over time the two liquids diffuse into each other. Provided that A is less soluble in the mixed solvent, solid (hopefully crystals) will form close to the interface of the liquids [3,4].

The denser liquid must be placed below the lighter and some exploratory experiments to find suitable solvents may be required. There are a large number of possible combinations with different solvent systems, solute concentration and temperature. The system is normally sealed to avoid solvent loss by evaporation and the tube in which the experiment is performed should be narrow relative to its length. You can set up several experiments and place them in the same screw-top bottle. Although the crystals will form at the interface (asuming they form at all) rough handling may cause them to fall to the bottom of the tube.

Unlike vapour diffusion, the solvents can be volatile or nonvolatile. Setting up requires care to layer the upper liquid on the lower. An ingenious method of putting the denser liquid in second via a pipette has been proposed and is shown in Fig. 2(b). Introduce the pipette and then introduce the denser liquid; finally carefully withdraw the pipette [9].

Freezing the (denser) lower liquid before adding the upper and then allowing the solid to slowly melt can also work.

  
Fig. 2(a) A typical arrangement. Fig. 2(b) Method of layering.
  


Slow evaporation

Here a solution of the compound is concentrated by evaporating off the solvent normally at a fixed temperature [4]. The solvent must be sufficiently volatile for the experiment to take a reasonable time and may disperse to the atmosphere or an absorption medium in a closed vessel (e.g. Removing water with a dessicant in a dessicator).

A crust is prone to form (in my experience) but given fairly large amount of solid can work. Crystals should be harvested before the system goes to dryness as dried crystals stick together and to the glass surface. Mixed solvents can be used but the relative evaporation is difficult to control/assess. You can try a mixture of solvents in which the solvent which dissolves the sample better is the more volatile.



Slow cooling

If the solubility decreases with decreasing temperature, then cooling a saturated solution will deposit solid on lowering the temperature. The rate of cooling must be slow to produce large crystals and a simple way to achieve this for temperatures < 100°C is to use hot water in a Dewar vessel and suspend the sample container in the water.

Accurately controlled cooling is more difficult to achieve and requires an electrically controlled heater-programmer. The surface of the solution should be covered to avoid contamination by dust etc. The freezing compartment of the 'fridge or freezer can be used to work below ambient temperatures. In the literature the use of mixed solvents is common to grow crystals.

One strategy we have used is to dissolve the compound in a solvent at room temperature; add second solvent to precipitate solid until opalescence starts; warm to dissolve; place in freezing compartment of 'fridge or freezer; cross fingers and hope.



Growth from vapour

Where compounds can be sublimed without decomposition, another method of both purification and crystal growth is available. Slow sublimation over several days gives larger crystals and typical arrangements are shown in Fig. 3. The system is often evacuated with static (tap closed) or dynamic (tap open) vacuum.

The crystals form on the cooler parts of the apparatus usually a few centimetres from the heater and the cool part may need to be kept cold with for example cold water. For very volatile materials it may be necessary to keep the cooler areas cold with solid CO2 slush baths or other refrigerants.

If left long enough, the daily and seasonal fluctuations in temperature can form crystals from a powdered material. I have grown nice crystals of PBr5 kept in a sealed glass ampoule by simply ignoring them for 20 years but this does seem extreme. (I confess - they were forgotten).

Sublimed crystals can adhere firmly to the glass walls and one method to dislodge them is to shock cool the glass in the region of the crystal using liquid nitrogen [28,29].

Crystals can be grown by sublimation directly in the capillary to be used for the X-ray diffraction experiment [30].

Examples of crystals grown by this method include: MF5, M = Mo [28], Ru [29], Nb or Ta [30].

  
Fig. 3(a) Typical horizontal arrangement. Fig. 3(b) Typical vertical arrangement.
  


Growth from gels

This method consists of allowing two materials (A and B) to react in a gel medium forming a solid that crystallises within the gel. One reactant can be included in the gel and the second in solution in contact (Fig. 4(a)). The alternative is for the two solutions containing the reactants to be separated by the gel (Fig. 4(b)). The reactants diffuse into the gel and over several days/weeks precipitate the product.

For example, copper crystals can be made using a reducing agent in a solution in contact with a silica gel containing Cu(II) ions [15]. The crystals formed have to be extracted from the gel which can be messy. Silica gel has been most widely used but organic gels have received some attention. Hanisch [20] is a widely quoted reference but other articles describe the method [15,21].

Examples of crystals grown by this method include: Cu [15], calcium tartrate [22].

  
Fig. 4(a) Gel experiment. Fig. 4(b) Gel experiment.
  


Thermal gradients

For fairly insoluble compounds, whose solubility increases with increasing temperature this method has found some success. It depends on convection taking the saturated (hot) solution and depositing solid at a colder part of the system.

It lends itself to the use of seed crystals but has been used for X-ray sized crystals. Some described systems are shown in Fig. 5 [8,27,31]. The triangular apparatus described by Hope [8] is ca. 10 cm tall and uses heating wire or tape around the sample and left-hand side 'leg'. Cooling the horizontal tube is done with water circulating in plastic tubing wound round or a copper heat sink. Crystals are said to form in the cool right-hand side 'leg'.

A small-scale version using a capillary has been described [25].

Examples of crystals grown by this method include: [Pb2I4(C5H5N)4] [27]

  
Fig. 5(a) Simple tube [31]. Fig. 5(b) Tube with side arm [27]. Fig. 5(c) Hope apparatus [8].
   


Hydrothermal methods

For aqueous systems of poorly soluble materials, hydrothermal methods may help. The drawback is the cost requiring pressure-sealed metal containers and temperature controlled heating ovens. These techniques find many industrial applications including quartz and the coloured varieties which are semi-precious stones used in jewelry (eg amethyst (violet)). Details of technically important crystals are available [33] and Hulliger [7] gives a few details/references to the method. See also Rabenau [35] for background to the hydrothermal method and use in the synthesis of inorganic substances.

Hector et al. [34] has used a teflon lined autoclave to prepare a number of iodate crystals by decomposition of metal periodates in the presence of periodic acid in water at 140°C. This rapidly yielded mm size crystals of materials that are difficult to prepare otherwise.

Searching the IUCr journals titles/abstracts (Acta Crystallogr. etc) for 'hydrothermal' produced a few 'hits' per year and this could be a good starting point if you wish to explore this method.



Biochemical methods

The biochemists have been well served by books [11,16] and reviews [12,13,14,19] on crystal growth particularly by McPherson. Although the biochemist is restricted to aqueous solutions, the parameters that can be adjusted include sample concentration, precipitant concentration, pH and ionic strength. Since suitable crystals only form in a small region of multi-dimensional parameter space, a large number of trials have to be carried out.

The commonest method is the vapour diffusion technique as either the 'hanging drop' or 'sitting drop' method (Fig. 6) using a small (10 μl where 0.1 ml = 100 μl) sample.

  
Fig. 6(a) Hanging drop. Fig. 6(b) Sitting drop.
  

Transport of water from the sample drop gives rise to crystal formation and the water transport is driven by the difference in vapour pressure over the two solutions and will stop when changes in concentration make the vapour pressures the same. Because the volume of the reservoir is large compared to the 'drop' the reservoir remains essentially unchanged.

Because of the large number of trials required there have been a number of successful attempts to automate the process with up to 96 wells in a multi-well plate being used. The size of the drop has also been reduced to save on valuable material.

In addition the biochemists make use of dialysis to produce crystals where small ions, but not the biomolecule can pass through the dialysis membrane [23].

Many of the techniques developed by the biochemists both for crystal growing and manipulation after growth are applicable to the small molecule scenario. There are several commercial companies supplying chemicals and apparatus. See for example the Web site of Hampton Research in the USA.



Miscellaneous methods

A small collection that do not fit into other categories.

1. A variant of the liquid diffusion method where the two liquids are immiscible was used to prepare Cd(H2O)2Ni(CN)4.4H2O from CdCl2 in the upper water layer and a salt of Ni(CN)42- that was soluble in the lower CH2Cl2 layer. Crystals formed at the interface [31].

2. Single crystals of insoluble compounds can sometimes be grown by diffusion of two solutions which interact to form the solid. The rate of diffusion can be contolled using a glass frit between the solutions and an apparatus has been described that allows easy isolation of the product [26].

3. In a truly heroic study of S6N4Cl2 during which one series of experiments lasted 12 months (temperature cycling), a vapour transport experiment yielded crystals. A horizontal sealed tube containing liquid SO2 and S3N2Cl2 was placed in a steep temperature gradient (+35°C at the hot (warm) end and -8°C at the cold end) yielded crystals on the upper surface (i.e. not in the liquid SO2). Clearly these experiment are dependent on the chemistry and not generally applicable [24].



Serious difficulties

If none of the methods seem to be leading anywhere the possibility of preparing a similar compound could be looked at. For example, with organic compounds use a different functional group whilst retaining the part of the molecule of interest. For salts, try using alternative anions (PF6-, BF4-, ClO4- (care!), BPh4-) or cations (NR4+, PR4+, AsPh4+, [N(PPh3)2]+) while retaining the counter-ion of interest.


Crystal size

In our crystal growing efforts what size crystals are we aiming to produce? Resisting any comments about 'size matters', the considerations are: the collimator size fitted to the diffractometer, the linear absorption coefficient for the crystal, and being able to record a satisfactory set of diffracted intensities.

The background to the diffraction experiment requires a uniform intensity beam of X-rays with the crystal lying completely in the beam in all orientations. The X-ray generator focal spot size and the choice of collimator determine the size of this uniform (circular) region at the crystal. More recently has seen the introduction of X-ray mirrors to focus the beam. A typical collimator size is 0.5 mm but smaller and larger diameters are used but < 1 mm. This sets an upper limit to the crystal size. You should check with the diffractometer operator/maintainer for the upper size of the crystal.

X-rays are attenuated on passing through solids and this reduction in intensity is governed by Beer's law:

I = Ioe-μt
where I, Io are the intensities of the final and initial beams, μ is the linear absorption coefficient and t is the sample thickness.

The linear absorption coefficient can easily be calculated knowing the wavelength of the radiation, the chemical composition of the crystals and the density.

The trade-off we have to balance is between a small crystal - weak scattered beams, low absorption and large crystal - strong scattering, high absorption. It is easy to show [see for example ref. 32, page 93] that an approximate optimum is given by 3/μ. [Stout & Jensen [4(a)] give 2/μ but we are in the same ball-park].

Thus for a weakly absorbing solid, for example an organic compound with μ = 2 mm-1 the 'optimum' size is 1.5 mm, and clearly much too large for a typical collimator.

For a strongly absorbing solid say an inorganic with heavy elements with μ = 20 mm-1 the 'optimum' size is 0.15 mm, and would be OK for a typical collimator.

[Take care: both mm-1 and cm-1 are used for μ].

The final thought should be the absorption correction you are going to use. In routine structure determinations, an empirical absorption correction is commonly used. When μt_mid (t_mid is the 'middle' crystal size - see the CIF (Crystallographic Information File)) is > 3.0, the International Union of Crystallography (IUCr) data validation procedures used in the software 'checkcif' require/recommend an analytical absorption based on indexed crystal faces and careful measurement of the crystal size.

BIG crystal

"What do you mean my crystals are too big" [55]



Use of microscope

The small crystals that may be formed can be examined using a small hand lens [x8 is a useful magnification. Bring the lens to the eye and then adjust the crystal-to-lens distance until in focus]. With a little experience material may be rejected at this stage. If it looks promising with suitable sized crystal of a reasonable morphology, then examination with a microscope can follow.

Both binocular and monocular polarizing microscopes are used. For both you will need to know how to set them up i.e. focus, change magnification, set sample illumination and bring the 'analyser' into the (polarized) light path. The polarizing microscope is widely used in mineralogy and allows an examination of the optical properties of transparent solids. You will need to know about the optical indicatrix (joy) [17] to make use of the advanced methods, but of course it is perfectly possible to select a suitable crystal without this. With the analyser 'out' you can look for nicely shaped single crystals that are not aggregates of two or more crystals or obviously twinned (re-entrant angles). With the analyser 'in' (crossed polars) the field of view is dark and crystals may show extinction in which a normally light coloured crystal becomes dark (extinguishes) as the crystal is rotated on the microscope stage. This extinction occurs every 90° rotation and a single crystal should go completely dark at some orientation. This often occurs parallel to a crystal edge and may be related to the crystal axes (a, b, c). Not all crystals show extinction: for example cubic crystals are isotropic and remain dark in all orientations and some views of the higher symmetry crystal systems are also isotropic under crossed polars.

Air-sensitive crystals can of course be examined in a glass ampoule or vial: they do not have to be placed on a microscope slide. Similarly crystals can be examined in mother liquor and indeed it is better to do this to avoid potential loss of solvent molecules from the crystals. Looking at your crystals under the microscope is a quick and useful procedure.

There are a number of books giving full details [1,18] and chapter 2 in the book by Dent Glasser [17] seems a good introduction to optical crystallography.



Toolkit

There are few things more irritating than finding in the middle of a difficult task that an essential tool is missing or that a consumable has been fully used up. I found this to apply to manipulating crystals under the microscope and preparing small-scale crystallisations as well as the task of mounting crystals for the diffractometer.

McPherson in his book [12] advocates collecting together various small items for handling crystals and I have found this to be excellent advice. My case contained a number of partitions of various sizes in which were stored anything and everything that had proved useful and if items were missing I had no one to blame but myself.

A extensive list would not be appropriate as technology moves on but surely any toolkit would include: microspatulas; dissecting needles and blades to select/cut crystals; an assortment of microscope slides (with/without depressions); Pasteur pipettes and teats; adhesives; greases; spare goniometer mounting pins; hand lens; ....



Crystal mounting

Although not part of the crystal growing methods, the manipulation and mounting of crystals for X-ray examination is the next step and a summary is given here.

There are several techniques for mounting your selected crystal on the mounting pins. The choice depends upon the data-collection temperature and the crystal stability. A good account is provided on the Blake crystal-growing website and several articles discuss particular methods but the text book treatments are usually brief.

There are also several good reviews on macromolecular cryocrystallography which contain comments useful for the small-molecule crystallographer [37,47,52,54,48].

It is important to know the height that the crystal should be above the top of the mounting pin in order to be in the X-ray beam. Modern goniometer heads have a limited adjustment - your crystallographer will let you know.

If you have air-stable crystals and are selecting under a microscope, a very thin smear of silicone grease on the microscope slide stops crystals - usually the best ones - flying off the slide when being trimmed. Hampton Research offers a range of very small tools for manipulating crystals but dissecting needles and sharp small blades (eg razor blades) usually suffice.

There are several well-known advantages to be gained from low-temperature data collection including reduced thermal parameters (atomic displacement parameters) and an increased intensity for larger sinθ/λ reflections [40,41,46,56]. Low-temperature working has become routine with the development of commercial equipment but this was not always the case and the road to the present position has been summarised [36,37]. Several articles have usefull practical comments to minimise the risk of ice formation [36,37] and the Garman and Schneider review [37] although aimed at macromolecule work is thoroughly recommended.

A number of laboratories seem to have adopted for small-molecule work the area-detector diffractometer/low temperature combination as the default.

The final point to remember is that for a (small) percentage of crystals, lowering the temperature can result in a structural change; this may be a reversible phase change or conversion to a polycrystalline material and a failed data collection. In such cases a room temperature data collection may be the answer.

Glass fibre mounting

For air-stable crystals the glass fibre mount can be usefull. The crystal can easily be stored for later use and data collection can be at ambient or low temperatures. It involves glueing the crystal to a short length of glass fibre which in turn is secured to the mounting pin (Fig. 1(a)). Various adhesives have been used over the years - they must securely anchor the crystal to the glass.

The glass fibre must be of small diameter relative to the crystal size and the crystal must be positioned so that it does not interfere with the incident or diffracted beams in all orientations during data collection. Scattering from the glass and adhesive can contribute to the background and should be minimised.

You should allow enough time for the glue to fully set or the crystal may change orientation during the experiment and invalidate the data collection.

For very small crystals a two-stage glass fibre is used with a short length (few mm) of very thin fibre from glass wool glued at the top of the conventional thicker fibre. The crystal is then glued to the glass wool fibre. Care must be taken that the crystal does not move relative to the goniometer head in different orientations due to the thinness or length of the mounting fibre or due to the gas stream if a low temperature device is in use. Blake has a useful discussion of glass fibres and more complicated arrangements.

People have also used glass capillaries as a replacement for the (solid) glass fibre as these are more rigid for the same mass of glass.

In the past workers have used diluted solvent based glues to coat the crystal and prevent slow decomposition but it is doubtfull if these would find many current applications.

[It is easy to produce metres/yards of glass fibres from a short length of glass rod (few mm in diameter) and a glass-blowing torch - it depends on how long your arms are! The fibres will come in all diameters but suitable ones can be chosen for the job in hand].

Glass capillary mounting

The use of thin-wall glass capillaries for both small-molecule and macromolecular crystallography is well established and has the advantage that room temperature experiments are possible.

Where the crystals lose solvent or are moisture/air sensitive a capillary mounting may offer a solution if a room temperature data set is required. Introducing crystals into the fragile capillaries is difficult particularly in a glove box and a holder consisting of a glass tube in which the wide end of the capillary sits supported by a ring of mouldable non-setting wax can save many broken capillaries (Fig. 7(b)). Introducing a film of silicone grease inside the capillary after the crystal has been introduced is a valuable method of stopping the crystal moving. The grease is introduced using a glass fibre smeared with the grease and inserted in the capillary. It is also possible to introduce a small amount of solvent and seal the capillary either by flame or an adhesive (eg rapid-set epoxy). See also [42].

Biomolecules contain large amounts of water and it is necessary to maintain the crystals in an atmosphere of the mother liquor. This is done by introducing liquid usually at both ends of the capillary and sealing (Fig. 7(c)) [49,50,51]. An alternative to the glass capillary has recently been proposed [57] in which the crystal on its mount is enclosed in a thin-wall polyester tube. The sealed tube also contains a small amount of stabilising liquid. Time will tell if this method is widely adopted but the authors suggest it has several advantages over glass capillaries.

Capillaries are commercially available with various internal diameters and made from various glass types or quartz. [See: Hampton Research and Hilgenberg GmbH, Germany (I'm not sure about the second company)].

The two following methods have markedly reduced the use of capillaries.

  
Fig. 7(a) Glass fibre mount. Fig. 7(b) Capillary holding device.
  
Fig. 7(c) Capillary mount. Fig. 7(d) Loop mount.
  
 

Oil-drop mounting

The oil-drop method was developed by Hope [36,47] and has found applications for both small-molecule and macromolecular crystallography. It is a low temperature technique and in the small-molecule case is particularly valuable for moisture-sensitive or reactive crystals. Some of the crystals under study are placed in a small pool of an oil sitting on a microscope slide. The oil acts to protect the crystals during selection. Hope used Paratone-N (Exxon) mixed with mineral oil [47] as the oil medium and Kottke and Stalke [41] in their procedure for reactive compounds used a mixture of perfluoro poly ethers (Hostinert216 and RS3000, (Riedel-de-Haën)). The proportion of the oils determines the viscosity and governs the temperature of the experiment. The use of Nujol, which can easily be dried, seems satisfactory in the 120-150 K range. After selection of the crystal it can be transferred to the end of a glass fibre previously secured to a mounting pin and rapidly positioned on the diffractometer and flash cooled in the gas stream.

The protective oil film also serves as an adhesive to hold the crystal in position. The same comments about the fibre and the position of the crystal with respect to the fibre apply as for the glass fibre mounting above. Perfluoro poly ether oils are available but the Riedel-de-Haën products given above may not be. (See Blake).

Hope has published detailed instructions for all the steps involved [52] and provided on the Web lecture notes from a 1995 meeting on cryocrystallography covering much the same ground.

A variant of the Hope method has been described for macromolecules avoiding the use of cryoprotectants and commenting on various oils [53].

Fibre loop mounting

The loop method was proposed for macromolecular crystallography by Teng [43] in 1990 in which a circular loop of thin wire is glued to the tip of a thin glass tube which in turn is attached to a metal mounting pin (Fig. 7(d)). The loop is typically 1-2 mm in diameter. A drop of (aqueous) solution containing the crystal can be scooped out of solution and the liquid held in position by surface tension. Cryoprotectants are selected so that on flash cooling a glass is formed in which is embedded the crystal.

This technique has been widely adopted by macromolecular crystallographers [37] and several methods have been described for constructing the loops from a variety of materials [44,45]. Each laboratory seems to have developed minor variants.

Hope mentioned in reference 2 the use of the loop method with an oil (rather than the normal aqueous medium) for very thin crystals.



Crystal growth at low temperatures

Growing crystals of compounds that are liquids or even gases at room temperature raises the level of technical difficulty and transferring these (hopefully) single crystals to the diffractometer provides yet more hurdles to be overcome. A second class of compounds that has been successfully studied are reactive chemical intermediates which can sometime be prepared as crystals in the low-temperature syntheses. All these studies are not routine experiments and specialized equipment is needed.

There are several reviews of this area which are briefly described below followed by a few examples which caught the reviewers notice. If you are not impressed by these or other examples you come across then you are very hard to please or have never tried the experiments yourself.

The Veith and Frank [40] review gives details of crystal growth in capillaries and a useful list of earlier studies of low temperature crystallisation and crystal transfer. Luger [39] gives details of crystal growth in capillaries for a range of low melting-point organic molecules. The apparatus described by Kottke and Stalke [41] allows the isolation and manipulation of low temperature crystals under a protective atmosphere and immersed in oil (perfluoro poly ethers). The selected crystal is then mounted on a fibre using the oil-drop method pioneered by Hope [47,36] and transferred to the diffractometer. More details of the Kottke and Stalke apparatus and an impressive range of organometallic intermediates that have been studied are described by Stalke [38].

A couple of examples:

1. The crystal of structure of AsCl5 was shown to be a trigonal bipyramid. Arsenic pentachloride does not exist at room temperature, decomposing to the trichloride and elemental chlorine. The antimony analogue does exist as a liquid at room temperatures and this was shown in single crystal X-ray studies to be a trigonal bipyramid above 219 K and a chlorine bridged dimer below this temperature.
[Ref: S. Haupt and K. Seppelt, Z. Anorg. Allg. Chem., 2002, 628, 729-734].

2. The lithium alkyls are highly reactive and important reagents in synthetic chemistry. They are also pyrophoric especially ButLi which has a m.pt. of -38°C. The crystal structure of this consists of a tetrameric Li4 unit with approximate Td symmetry with each face capped by a t-butyl residue.
[Ref: D. Stalke, Chem. Soc. Rev., 1998, 27, 171-178].



Tales of woe

We are not concerned here with the formation of solvates during the crystallisation process which today is usually a minor inconvenience although taking a disproportionate amount of time to model if disorder is involved. [It was more of a problem before routine low temperature data collections as avoiding loss of solvate meant sealing the crystal in a capillary possibly with traces of solvent]. Nor are we dealing with the times that the crystals lovingly produced (I exaggerate) turn out to be the wrong (but related) compound.

The commonest category is when an unexpected element is present:

1. The compound MoCl2(PMe3)2 based on an X-ray crystal structure turns out to have been ZnCl2(PMe3)2. The Mo (Z = 42) and Zn (Z = 30) problem might have been spotted in the thermal parameters in the original work and as pointed out in the correction, chemical analysis would have raised doubts (eg Mo: C=22.59%. Zn: C=24.98%). Even an unfashionable (but quick) density measurement (what are they?) shows differences between the calculated values for Zn (1.363 g cm-3) and Mo (1.507 g cm-3) (based on the room temperature cell).
[Ref: F. A. Cotton and G. Schmidt, Polyhedron, 1996, 15, 4053-4059].

2. The compound reported as TiOCl(acac) and formulated as a dimer was later shown by the crystal structure to be SnCl2(acac)2 where Hacac is acetylacetone. The tin was used in the preparation of the alleged titanium compound. This case illustrates where chemical analysis can mislead as both formulations have very similar C, H and Cl analyses.
[Ref: M. Webster and J. S. Wood, J. Chem. Res., 1981, (S), 40; (M), 0450].

3. In studying the reactions of TiI4 with the ligand diars [o-C6H4(AsMe2)2], crystal structures were obtained of two materials initially formulated as [TiI2(diars)2] and [TiI2(diars)2]I. At the time the chemical point that rang alarm bells was Ti(II) for the former and a follow-up EDX analysis showed that the crystal contained Fe and essentially no Ti. The batch of commercial TiI4 was also shown to contain Fe. Refinement as the Fe compound produces a better fit to the data. (Ti, Z = 22; Fe, Z = 26).
[Ref: W. Levason, M. L. Matthews, B. Patel, G. Reid and M. Webster, Polyhedron, 2004, 23, 605-609].

4. Your beautiful single crystal may not be a single chemical species (!) and for a time this problem created a flurry of activity. MoOCl2(PR3)3 (cis/mer) exist in two forms with different colours (blue or green) and the Mo-O distance varied from 1.80(1) Å in 'green' (PR3 = PEt2Ph) to 1.676(7) Å in 'blue' (PMe2Ph). In brief the crystals contained MoOCl2(PR3)3 AND MoCl3(PR3)3 with the atoms occupying essentially the same position in the cell apart from the O and Cl. The "Mo-O" distance was an average over the collection of unit cells of the Mo=O (1.7 Å) and Mo-Cl (2.4 Å) distances. This was the origin of the so-called "bond-stretch isomerism".
[Ref: G. Parkin, Acc. Chem. Res., 1992, 25, 455-460. G. Parkin, Chem. Rev., 1993, 93, 887-911].

5. Although the reaction vessels are normally regarded as unreactive, in at least one example the desired compound, a potassium organoindium species, reacted with silicone grease during the crystallisation to give a product containing the [K(Me2SiO)7]+ cation.
[Ref: M. R. Churchill, C. H. Lake, S.-H. L. Chao and O. T. Beachley, J. Chem. Soc., Chem. Commun., 1993, 1577-1578].

6. Glass is normally unreactive but under some conditions atoms in the glass can be leached out into solution and finish up in crystals. During attempts to prepare crystals of a platinum periodate complex, H2PtCl6 and KIO4 were added to aqueous KOH and the mixture heated to 140°C in a sealed glass tube for a few hours. The resulting crystals were found to contain Na as well as K presumably arising from attack of the glass surface. The M...O distances in the crystal structure were too short for M to be K and more in keeping with Na and this was confirmed by EDX measurements on the crystals. Earlier attempts to grow crystals of the K salt had failed but in the presence of K and Na cations suitable crystals were isolated - a happy accident.
[Ref: W. Levason, M. D. Spicer and M. Webster, J. Coord. Chem., 1991, 23, 67-76].

7. Another reaction where glass provided an unexpected species is the report of a PF6- anion in a crystal structure which is now believed to be SiF62-, based not on any crystallographic arguments (bond lengths, atomic displacement parameters, etc) but a simple charge balance requirement.
[Ref: Anon.].

8. In the structure of the dioxide derived from the diphosphine ligand o-C6H4(PPh2)2 [(1,2-diphenylphosphino)benzene] one of the -P(O)Ph2 groups had an abnormally short P–O bond (P1–O1) and the O1 atom a large anisotropic adp ellipsoid. The R-factor was a reasonable 0.054 (R1 in Shelxl) and nothing else seemed out of order. The model put forward was of a partial O1 atom (site occupation factor = 0.4) giving adp values for the two O atoms similar values, an improved R1 (0.038) and an explanation for the short bond. In the chemical oxidation of the phosphine, incomplete reaction had occured and the crystals contained both monoxide and dioxide with the atom sites being nearly identical (apart from O1).
[Ref: M. Webster, unpublished, 2004].

Is there a moral to these examples? Probably not, but no one likes making mistakes particularly in public and a little sympathy might not be out of place. Of course, a number of the above examples were picked up during the work and corrected, but for the others hindsight is a wonderful gift.

Perhaps the main points are to endeavour to retain an open mind about your results and to keep an eye open for things that don't seem quite right.



References

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These notes were assembled in the mid 1990s as part of a handout to postgraduate students in the Department of Chemistry, University of Southampton, UK. Later revised they formed part of a booklet for a P/G course on physical methods which has been discontinued. They have been revised and extended in this web version.
Michael Webster.  October 2003.
Last update: November 2005. V1.3
Material Copyright © 2003-2005
  Feedback is welcomed. Suggestions for improvement, reporting of errors, etc should be sent to: Dr Mike Webster (email: mw1@soton.ac.uk).