Crystal growth of inorganic, organic, and biological macromolecules in gels

Progress in Crystal Growth and Characterization of Materials xxx (xxxx) xxx–xxx

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Crystal growth of inorganic, organic, and biological macromolecules in gels Abel Morenoa, María J. Rosales-Hoza,b,* a b

Instituto de Química, Universidad Nacional Autónoma de México, México D. F. 04510, México Departamento de Química, Centro de Investigación y de Estudios Avanzados, Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, México D. F., México

“Nowadays, all we know is that Nature takes thousands and thousands of years to obtain beautiful minerals (gems) like sapphires, rubies, diamonds, among others that have made people crazy due to their beauty, but the gel method was born to allow us to play with instantaneous mineralogy” H.K. Henisch 1988 [1]. 1. Historical data on the gel method The pioneering experiment related to crystal growth in gels was performed in 1896 by Liesegang, who obtained periodic precipitation of inorganic compounds grown in gelatine [2]. These rings were considered of interest, partly due to their obscure origin and partly because they were reminiscences of certain structures found in nature [2]. A year later, in 1897, Wilhelm Ostwald was also the first to propose an explanation to the phenomenon observed by Liesegang [3]. Ostwald's hypothesis was based on the propagation of a supersaturation wave; this suggestion was matured by Ostwald up to the middle of the 1920’s [4]. At that time, there had already been scientific contributions that attempted different crystal growth experiments such as ice crystals grown on ice cream, Rochelle’s salt grown in cheese, and crystals of sulphur grown in rubber [5]. There were some publications even on the crystallisation of uric acid in joints [6]. However, all these experiments were inconsistent according to the principles of crystals growth in gels in terms of reproducibility. The gel method was neglected just until the beginning of the 50’s of the twentieth century, when a PhD student from Harvard University (Lewin, 1950) first attempted to crystallize biological macromolecules in gels. At that time, most geologists thought that all quartz on earth had once been silica hydrogels. The most remarkable experiment closely related to the crystal growth in gels was performed by Eitel in 1954 [7]. According to historical records, microscopic crystals of silica were obtained from silica gels in the presence of various “crystallisation agents” when heated in water vapour under certain pressure. In 1954 Low and Richards reported, for the first time, the crystallisation of albumin in gelatine (based on the idea of Lewin [8]. Then, there was, once again, a gap (with very few reports of the use of gel growth method), up to 1975, when organic gels were introduced for biological crystallisation in a short review published by Dondi [9]. He applied the gel method for electrophoresis in the crystallisation of ribosomes in order to crystallize these large biomolecules in situ. *

During the 70’s and the 80’s all information related to the gel growth method was summarized in the book published by Prof. Henisch in 1986, including some chapters related to theories of crystal growth and transport processes that were co-signed with Prof. García-Ruiz [10,11]. This book inspired many scientists to grow crystals (mostly minerals, but also some organics) in different types of hydrogels and a variety of experimental setups (see Figs. 1 and 2). The French group focused on mineral growth led by Lefacheux who introduced in 1988 the use of specific silica gels (tetramethyl orthosilicates) in the crystallisation of proteins and inorganics. Even this group, suggested agarose as one of the easy-to-use gels for the growth of crystals of any kind [12]. At the beginning of the 90’s in the twentieth century, the group of Prof. García-Ruiz, in Spain, pioneered a theory involving molecular simulations of crystal growth in gels as diffusing-reacting systems [13]. He also proposed to use gel media to crystallize inorganics and biological macromolecules (proteins, nucleic acids and polysaccharides) by using the gel-growth approach [14]. Another approach closely related to crystal growth in gels includes the possibility of crystallising proteins in capillaries (with a diameter less than 0.3 mm). A development of the gel-growth technique involving the role of reducing convective transport as occurs during crystal-growth in capillaries or in microgravity experiments are closely related strategies for the production of highquality single crystals. Additional consecutive advantages of minimising convective transport has been demonstrated using the capillary tube method [15]. These advances in the growth of proteins in capillary tubes permitted Prof. García-Ruiz and his team to develop, in 1993, the first variant of the counter-diffusion methods, called the gel acupuncture technique (well-known with the acronym GAME) [16]. This novel technique consists of a precipitating agent that diffuses through the gel inside a capillary tube filled with a protein solution, thus also enabling crystallisation of small molecular weight compounds [17]. These methods applied to proteins will be discussed at the end of this review. Nowadays, the gel growth technique (Fig. 1) has undergone some innovations that includes the use of ad hoc devices (Fig. 2) used to grow different types of crystals. This technique has been categorized as part of counter-diffusion methods thanks to the assessment of the different types of gels, capillaries, additives, and different types of crystal growth devices used for the crystallisation of different compounds from small

Corresponding author. E-mail address: [email protected] (M.J. Rosales-Hoz).

http://dx.doi.org/10.1016/j.pcrysgrow.2017.04.003

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Please cite this article as: Moreno, A., Progress in Crystal Growth and Characterization of Materials (2017), http://dx.doi.org/10.1016/j.pcrysgrow.2017.04.003

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Fig. 1. Basic procedures to grow crystals in gels. a) method two layers, b) method three layers, c) U-tube method.

Some reviews recently published have demonstrated the potential of the gel-method for producing high-quality single crystals of a variety of compounds and biomolecules, compared to crystal growth in solution [23–25]. This gel method was already known at the end of the nineteenth century, however, it had not been widely used to grow crystals from many different compounds until now.

to biomacromolecular crystals [18]. In contrast to other techniques using capillaries, different levels of supersaturation can exist, allowing the precipitation to occur in very high supersaturation zones (nucleation occurs when supersaturation is high, and growth occurs as supersaturation diminishes). Several authors have demonstrated that gel growth increases the quality of crystals as compared to solution growth as well as demonstrating the probability of finding optimal crystallisation conditions (see for instance reference [15]). So far, we have generally mentioned the advantages of the gel growth method of inorganics, organics and soluble proteins. However, there are also some strategies and approaches known as in meso crystallisation techniques applied to membrane proteins, (including crystallisation in lipidic cubic and sponge phases) [19]. These techniques have allowed us to obtain over 250 structures of membrane proteins (of these 250 structures over 80 represent unique proteins [20]). The in meso techniques could be considered as one of the approaches to crystal growth in gels, they use lipidic or sponge phases instead of gel network. This latter assumption is due to fact that the principles of diffusingreacting systems and diffusion transport control are perfectly applicable to this in meso techniques [21,22].

2. Type of gel used to grow crystals: synthesis and structure Several types of gels have been used for the crystallisation of different types of molecules. The structure of gels where atoms or molecules can diffuse, favours the preparation of good quality crystals. The solubility and the pore size need special conditions of pH and temperature. These determine the type of gel to be used for a certain type of sample. The materials used in crystal growth include agar, silicates, oleates and even fruit jelly [26]. In recent experiments poly(ethylene) oxide has also been used for the crystallisation of inorganic and organic molecules in non-aqueous solvents [27]. The great majority of reports describing crystallisation of different compounds in gels, have used either agarose or silicate hydrogels. Fig. 2. Device based on the U-tube method. This method allows compounds A and B to counter-diffuse and obtain crystals in the central part.

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growth [35]. In the specific case of crystallisation in gels, some attention has been paid to the effect of molecules present in nature on the biomineralisation processes of metallic salts, such as calcium carbonate. Among these additives we can find peptides or proteins. Some biocrystallogenesis studies involved an analysis of the crystallisation parameters of calcium carbonate on agarose viscous solutions by counter-diffusion, adding charged polypeptides [36]. The results showed that cationic polypeptide affected the crystal growth mechanism of calcium carbonate, whereas anionic polypeptides affected both the nucleation and growth processes. The fact that alginate has been observed to have a large effect on crystal growth rates of vaterite, can account for the small crystals that can be found inside the gel network in alginate beds [37]. More recently, pollution problems in different parts of the world have triggered the study of the effect of pollutants on mineral growth. The presence of a toxic pollutant such as Cr(VI) in the crystallisation medium might have an important influence in both the nucleation and growth of crystals of CaCO3. Some studies have shown that the presence of Cr(VI) promotes the formation of higher quantities of vaterite and aragonite in the presence of chromium rather than in its absence [38]. Cr(VI) also reduces the nucleation density of calcite proportionally. The studies also showed that an increasing concentration of Cr(VI) in the medium produced a decrease in the mean size of single crystals and aggregates, as well as a change in the habit of calcite crystals, although it increased the amount of incorporated chromium.

These two types of gels have proved to be efficient in the synthesis and grow of high quality crystals in aqueous media. The procedure is as follows: first we dissolve one reagent in the gel and then we add a dissolution of another reagent on the top of this gel. Experiments have been carried out using a wide range of variables such as the pH and temperature of the gel as well as the concentration of the reactants (here the reactants are dispersed at the temperature of the nucleation) and then crystal growth processes take place. The fact that the crystallization in hydrogels such as agarose and silicates are extensively used, is due not only to the fact that they provide a successful media to grow high-quality crystals of proteins and of inorganic salts, but also to their interesting model system that mimic the formation processes in biological systems for the preparation of biomorphs and/or biological materials (see the last part of this review). The formation of gels depends clearly on the type of gel’s synthesis process (acidification, alkalinisation, hydrolysis, condensation, etc.). In the case of silicate gels, polysiloxane gels are the result of either neutralisation reactions (if sodium metasilicate (SS) is used) or polycondensation reactions (if tetramethyl orthosilicate (TMOS) or tetraethyl orthosilicate (TEOS) are used as gel ’s precursors) of silicate ions, which leads to the formation of a dense network of silicon-oxygen arrangements in the structure of flexible polymers. Agarose consists of long chains of a polysaccharide which associate to form double helices, which then bundle together with many van der Waals interactions between chains. Agarose is a physical gel because the association is reversible with heat. However, silicate gels are classified as chemical gels, because the interatomic interactions form covalent bonds. Chemical gels are irreversible in the process of forming the liquid precursors after changing temperature as happens with agar [28]. One of the most important properties of gels is the pore size (this depends on the type of gel and the conditions in which it was prepared). Sodium silicate, tetramethyl orthosilicate and tetraethyl orthosilicate show pore sizes that range from 50 to 250 nm with TEOS showing the largest sizes [28]. The size of crystals is generally related to the size of the pore, however, there are also other factors that can affect the size of the crystals. The characterization and the determination of the pore size had not always been easy in the past. Some of the first images of the pores, were obtained by Moreno et al., at the end of the 90’s [29] by scanning electron microscopy (SEM). This research showed that TMOS and TEOS gels structure could be characterized by small angle X-ray scattering (SAXS), and atomic force microscopy among other techniques [29]. The crystallisation of a given compound in different types of gels, can produce crystals with different morphologies. A study on the crystallisation of sodium bromate in agarose [30], silica and gelatine gels, show that different crystal habits can be obtained, by controlling solute concentration and gel density. Other studies [31] showed that several inorganic salts changed their morphology from a polyhedral shape into dendritic forms as the gel density increased, regardless of the type of inorganic material or gelling agents used. It is important to point out that it is possible to obtain several polymorphs of a compound in the same gel, and even in the same experiment (as observed in the crystallisation of lead phosphate in a polyacrylamide gel) [32]. The presence of electronegative groups in both silica gels and agar, can induce strong electrostatic interactions with the active molecule that is being crystallised. These interactions promote the incorporation of the gel into the crystal producing crystal composites. These composites have different solubility depending on the strength of the electrostatic interactions [33]. Agarose gels can also be incorporated into the crystals, mainly because of the fibrous nature of the agarose gel’s network [34]. Another aspect that recent studies have considered in some detail, is the effect of additives in the crystallisation process. It is well-known that the presence of additives exerts an influence on the different aspects of the crystallisation process either in nucleation, or crystal

3. Organic molecules crystallized in gels In comparison to the extensive work carried out on the crystallisation process of large biomolecules in gels, not much has been done regarding smaller organic molecules. This is partly due to the fact that the most widely used gel crystallisation procedures involve hydrogels and both solubility and stability problems have precluded the use of gels. Furthermore, the crystallisation procedures involving slow evaporation of the solvent in concentrated solutions, have given good results in many cases [39]. However, some compounds have been successfully crystallised in water based gels. Crystals of very good quality of molecules on biological interest such as steroids, have been obtained using these methods. Such are the cases of testosterone [40] and cholesteryl acetate [41]. Sodium methasilicate was used as the gelling principle in the testosterone case. The gel was prepared using water-acetone solutions and the pH adjusted to 5 to lead to the formation of good crystals of the hydrated form B of testosterone. Similar conditions were used for cholesteryl acetate. During the diffusion of this last compound into the gel, its solubility was reduced which led to supersaturation of the compound in the gel, causing nucleation and crystal growth. Aminoacids are other molecules of biological interest whose crystallisation processes in gels have been studied. A study on the crystallisation of asparagine monohydrate in different media yielded crystals with different morphologies [42]. Some of these results have been proposed to be due to interactions between the crystallising solute and the growth media. The authors of this study point out that “the study of molecular crystal growth in gel media is an underdeveloped area that provides a means to generate crystals with some unusual characteristics.” The crystallisation of glycine, the smallest aminoacid, in gels, has also been attempted [43]. Three types of crystals of glycine have been described: alpha, beta and gamma. The last form crystallises in the noncentrosymmetric space group P31, a fact that has attracted attention for his potential applications in non-linear optics [44]. The least stable form is the beta one. Crystallisation in silica gel produced the alpha form, and, at pH 4, the gamma form. The quality of these crystals was better than that of crystals obtained from aqueous solutions. Other crystals with potential applications in non-linear optics that were successfully grown through gel-solution techniques were methyl3

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cyclodextrins are soluble in water, but insoluble in alcohols. We tested the influence of low and high the molecular weight (MW) alcohols and found a very interesting trends. For beta cyclodextrin the higher the molecular weight of the alcohol used for crystallisation the smaller the crystals. On the other hand, for gamma cyclodextrin the higher the MW of the anti-solvent (alcohol) diffused in gels, the larger the size. These experiments are particularly interesting, because they show the importance of the anti-solvent, as well as the type of gel use for the crystallisation. Further experiments will be done in our group on this research, not only for characterising the structure of the cyclodextrins, but also the existence of certain polymorphs. Let’s move on with another example using a PEO gel. Good quality crystals of triiodo phenol were also obtained from a PEO gel using dichloroethane as solvent and an organic solvent as a precipitating agent (anti-solvent) [53]. Crystals with different morphologies were observed and successfully used for light scattering studies. More studies on the crystallisation of organic molecules using gel techniques are necessary in order to be able to determine the usefulness of this method on the handling of certain aspects of molecular structure such as chirality.

p-hydroxy benzoate [45] and 2-amino-5-nitropyridinium dihydrogen phosphate [46,47]. Crystal growth in gels has also been used in order to study polymorphism. An example of this is the study of ( ± ) − modafinil [( ± ) − 2 − ((diphenylmethyl)sulfinyl)-N-acetamide], an active pharmaceutical ingredient which crystallises in several polymorphic forms. Forms (I) and (III) have been known for some time [48]. The racemic form (I) is a the most stable one, while form (III) is metastable. For some time, form (III) could not be crystallised and this prompted studies of crystallisation to obtain good quality crystals of this polymorph. Crystallisation experiments in tetramethoxisilane (TMOS) gels yielded crystals that showed several morphologies. The already known form I was the first to be observed, but form (III) could also be obtained. Twinning of form (I) was observed but also packing that combines both forms. Dissolution of these last crystals showed that one body of the twin dissolved smoothly with a regular disappearance, the second body dissolved heterogeneously leading to lamellar fragments. Since form III is metastable, it is presumed that this is the form that dissolves faster [49]. More recent studies [50] have shown that an equimolar mixture of both enantiomers of forms I or III, in TMOS gel, lead to the formation of another polymorph: form (IV) which crystallises in the unusual Fdd2 space group. A comparison between the polymorphs shows clear differences in conformation and packing of the molecules. The examples of crystallisation in gels presented so far, have been achieved using hydrogels. There is, however, a need for processes that allow crystallisation of samples that are unstable or not soluble in water. That motivated the study of other gelling materials [51]. The use of poly (ethylene oxide) PEO, as a gelator, allowed the use of a variety of organic solvents [52] using different techniques. Experiments of solvent evaporation using a PEO gel allowed the crystallisation of 6chloro-2,4-dinitroaniline while through an antisolvent liquid diffusion in PEO using methanol as the antisolvent, crystals of beta-cyclodextrin were obtained. Other techniques such as the use of crystallisation mushrooms that allowed a vapour diffusion process in nitromethanes, lead to the formation of good quality crystals of acetaminophen. In our group, further experiments of the crystallisation of beta- and gamma-cyclodextrins were carried out as we can see in Figs. 3–6. The

4. Inorganic molecules crystallized in gels A review of the literature shows there are more examples of crystals of inorganic molecules obtained in gels. The larger number of examples is found among derivatives of the main group elements. This is mainly due to the interest of comparing synthetic crystals with those found in nature in compounds such as calcium salts. Calcium is one of the most abundant elements on earth. It is widely distributed in minerals and in the sea. While on earth, it is mainly found as the mineral dolomite (CaCO3.MgCO3), in the oceans, an important amount is found forming exoskeletons of sea organisms. There are three main polymorphic types of calcium carbonate found in nature: calcite, aragonite and vaterite. At room temperature and atmospheric pressure, calcite is the most stable polymorph but aragonite constitutes 95% of mother of pearl [54], where crystals show there is control on the polymorph, the size and the organic-inorganic type of interaction, underlying the importance of the conditions in the crystallising process Fig. 3. Crystallisation of beta-cyclodextrine in: a) methanol, b) ethanol and c) isopropanol. The leftside corresponds to a conventional Pasteur pipette.

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Fig. 4. Gamma cyclodextrine grown in PEO (100k MW) in Ethanol. These crystal were grown in a Pasteur Pipette as shown in the previous Figure.

crystal. The presence of proteins affects the crystal growth as does the presence of Cr(VI) or magnesium. In fact, experiments carried by García-Ruiz and co-workers, have shown that incorporation of silica from the gel into the crystal arrangement of calcium and other alkaline-earth carbonates, to form dendritic, sheet like and spiral forms [60–62]. Further studies [63] on the crystallisation of strontium carbonate, indicate that when crystals form within the gel, there is a reduction of the subunit size and a reduction of the growth direction by selective adsorption of silicate anions thus leading to the unusual morphologies mentioned. The case of strontium carbonate and other alkaline-earth carbonates should be studied in detail, as one of the most interesting case studies for crystal-growth, where we can see the effect of the type of gel to obtain single crystals or the formation of induced morphology crystalline aggregates, usually called biomorphs in the argot of the literature of these specialized studies. Fig. 8 shows the crystal growth of strontium carbonate in silica hydrogels (at alkaline pH) by using the experimental set-up shown in Fig. 1a. The component A was Na2CO3 in the gel layer and the second reactant (component B) was a solution of SrCl2 both in aqueous solution 0.1 M in concentration. The SrCO3 in agarose produced a dendrite-like crystals, while in sodium metasilicate hydrogel prepared at alkaline pH (8–10) produced biomorphs a kind of

(Fig. 7). The chemical composition in living organisms developing layers of calcium carbonate are quite complex. There are peptides and proteins present as well as other minerals in the environment. A study carried out in several organisms showed that amorphous calcium carbonate was mixed with important quantities of proteins that were rich in glutamic acid, serine, glycine and polysaccharides with proteins rich in aspartic acid seem to induce the formation of calcite crystals [55]. Analyses of samples of calcium carbonate taken from several organisms, have shown the presence of different gel systems such as alginate [37] or a proteinaceous sulphate-rich polymer that behaves as a hydrogel. In order to simulate nature, different experiments have used gels in order to provide a matrix that would serve as a scaffold for the growth of the salt particles. An understanding of the conditions in which these biomaterials develop, is very important for the synthesis of other materials. Therefore, several experiments have been carried out in which peptides [36], proteins [28,56,57] or other metal ions [38,58,59] are added. These experiments have shown that crystallisation in gels has an important effect in aggregate formation although no influence is apparent on the mineral orientation. Agarose and gelatine behave different from silica gels [59]. Hydrogels tend to get incorporated into the

Fig. 5. Crystal growth set up of gamma-clyclodextrine in agarose (for details see the text).

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salts, motivated the study of other derivatives. Such is the case of barium diphosphonates, molecules that have potential applications in catalysis and adsorption [66]. Different phosphonate compounds were prepared using different techniques in the same type of silica gel; in one case, both components of the salt were allowed to diffuse in the gel separately while in the other two cases, a solution with both components diffused in the gel together. The first case lead to the formation of a novel packing structure but since no comparison is described of the application of the same technique with the other two diphosphonates, it is not possible to say whether this was an effect of the experimental conditions used. Crystals of strontium tartrate were also successfully obtained in a silica gel and calcium tartrate as models of chemical compound for gel growth [67]. A control of pH and thickness and age of the gel allowed the formation of crystals that could be characterized by laser scattering tomography. One frequent problem in the crystallisation processes of both organic and inorganic compound is the low solubility they show. The gel method of crystallisation has been useful in some of such cases as it was shown in the case of PbHPO4 where diffusion of solutions of lead nitrate and phosphoric acid created a precipitation zone where there was a low concentration of lead nitrate and a high concentration of phosphate anions which allowed the crystallisation of PbHPO4 [68]. This method produced better quality crystals than other evaporation methods had allowed. Gel crystallisation has also shown its usefulness in the growth of unstable crystals which are explosive. Such is the case of lead azide, which has been shown to be present in two polymorphic forms; a monoclinic β-phase which is unstable and which transforms into the rhombic α-phase. The β-phase is also more sensitive to external stimuli with the possibility of exploding. Researchers have been trying to understand the changes of phase but the handling of the samples posed an important problem until a device was proposed where a gel was used as a crystallisation media and this allowed a much better control of crystal size and the danger of explosions [69]. Other reports of crystallisation of main group elements compounds [70,71] in gels, show the potential application of this technique for crystal growth of inorganic derivatives even when they have properties which present a challenge in their manipulation. Control of the crystallisation conditions might allow the preparation of a large number of crystalline materials not available up to now.

Fig. 6. Crystals of gamma-cyclodextrine obtained in a) methanol, b) ethanol.

precipitation described in references [62–64]. In the case of other inorganics, more recent studies [64] showed that the morphology of crystals of Ba(NO3)2, NH4Cl, H3BO3 and K2Cr2O7 changed from polyhedral habits into dendritic forms as the gel density increased. This occurs regardless of the organic gelling agent used (agar, gelatine, pectin or poly (vinyl alcohol)). This indicates that diffusion was the most important factor for crystal growth in a dense gel matrix because the organic molecules of the gelling material interact with the inorganic ions. Seeking a better understanding on the formation of crystal morphologies similar to those found in nature, the crystallisation of barium carbonate in silica gel was studied, analysing the effects of pH on the morphology and spatial formation of the crystals within the gel [65]. The necessary pH for these biomorphs to form, was found to be less alkaline than previously believed. The success in the preparation of crystals of several alkaline earth

Fig. 7. Crystallisation of calcite (CaCO3): a) in solution, b) in gels (agarose hydrogel), c) U-tube set up. The scale bars in a) and b) are the same.

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Fig. 8. This picture corresponds to SrCO3 synthesized in sodium metasilicate gel prepared at alkaline pH.

using PEO gels [52].

Even though much less work has been carried out with compounds of the d- or f-block elements, there are some reports on the use of gel crystallisation techniques for derivatives of these metal ions. For example, uranyl phosphate crystals have been obtained in a tetramethoxysilane gel [72]. Good crystals of this compound are difficult to obtain through the traditional slow evaporation techniques due to the low solubility of the salt but the use of the gel technique showed the potential application of this process for the synthesis of crystals of compounds containing rare earth elements. The formation of crystalline periodic patterns has also attracted attention in recent years because of the possible applications they can have in technological fields. It is known that the kinetics of crystal growth is very important to determine the morphology of the crystals formed. Sinuous and wavy patterns have been observed to occur with the use of polymer matrices for crystal growth. Such was the case in the crystals of copper sulfate pentahydrated obtained in a gelatine matrix [73,74]. The form of the grown crystal changes from polyhedral to irregularly branching morphologies as the polymer density was increased. In an effort to understand the phenomena occurring in a gel when two reagents diffuse trough a gel, copper sulphate has been studied in some detail. In the last few years, much attention has been paid to the development of supramolecular compounds. Among these supramolecular derivatives MOF’s (metal-organic frameworks) [75] and coordination polymers [76] have been extensively studied. Since parameters such as crystal nucleation, crystal growth kinetics and thermodynamics, are important parameters for the development of such derivatives, a study of the influence of gels was of interest. Several metal ions and simple but flexible ligands were mixed in gels in order to observe the influence of the media in the preparation of coordination polymers [77]. Experiments showed that controlling parameters such as concentration of the reagents, it is possible to prepare coordination polymers by the use of single gel diffusion techniques. Rare-earth coordination polymers have also been prepared using this technique. Several compounds can only be obtained by the use of gel crystallisation procedures [78]. Yaghi and coworkers prepared crystals of a zinc complex of 1,3,5.benzenetricarboxilic acid and pyridine with the use of PEO gels to yield a structure with a 3D-arrangement that contains square channels with pores of nearly 6 Å [79]. Some other coordination compounds have also been successfully crystallised

5. Biological macromolecules crystallised in gels and practical aspects 5.1. Crystallisation in gels and in capillaries containing a gel network Whatever the chemical nature of the gel, it should be neutral. In the case of agarose, pore size distribution depends upon its molecular weight and its degree of derivatization [80]. Charged groups may bind biological macromolecules. Very pure agarose gel is now available for protein crystallisation (Hampton Research). Agarose can be used with virtually all crystallisation methods: vapour diffusion, dialysis, freeinterface diffusion, and in capillaries [23,81]. Batches with a low gelling temperature (Tgel < 28 °C, commercialized e.g. by Sigma) are preferred to prevent the denaturation of heat labile macromolecules. At low concentration, the transparency of agarose allows one to monitor nucleation and crystal growth phenomena by optical methods. However, it must be mentioned that agarose does not form a gel in the presence of all precipitants, especially ammonium sulphate [18]. For this reason, it is safer to trap the protein in the gel at low ionic strength in the absence of precipitating agent. This will guarantee a well-defined and reproducible polymer structure and porosity [82]. The mechanical properties of agarose [83] make of it a good shock absorber during crystal storage and transport. Small temperature variations are also attenuated (Fig.9). On the other hand, polysiloxane hydrogels have not been used as frequently as the natural biopolymer agarose. There are probably two reasons for this; they are not currently available in biological laboratories and their preparation requires more handling. Further, unlike agarose soluble orthosilicate silicates (tetramethyl orthosilicate, TMOS and tetraethyl orthosilicate, TEOS) are not chemically neutral and should never be in direct contact with proteins. In addition, these liquids are not soluble in water [12]. Their gelling process involves a hydrolysis followed by a polycondensation which requires certain expertise in the gel’s preparation. Experimenters should be aware that the hydrolysis consumes water (vigorous stirring accelerates dissolution and clearing) and releases alcohol (methanol or ethanol, respectively). The molar ratio of water to TMOS is correlated with the size of the pores in the gel matrix [84]. For a TMOS gel of percentage p, the volume of water is 2.5 × p ml and that of methanol 10.9 × p ml, another point is 7

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Fig. 9. Crystallisation of protein crystals in capillary tubes and in gels. The left-side shows the crystallisation set up. Figure a) shows crystals obtained from solution and the right-side b) shows crystals obtained in agarose gel. The effect of the transport control in the crystal growth of thaumatin, a protein obtained from Thaumatococcus daniellii (African miraculous berry) can be appreciated. The diameter of the capillary is 0.5 mm in both cases. [11] [12] [13] [14]

that the high strength reached in concentrated TMOS or TEOS hydrogels may change the morphology of protein crystals [85]. 5.2. Crystal recovery from gels

[15] [16] [17] [18]

The major concern of the crystallographer is the recovery of crystals grown in gel. The situation is not as complicated even when the gel is enclosed in a capillary. Once the ends of the capillary are open, a little air pressure is sufficient to extrude the gel with the crystals in a drop of mother liquor (containing all ingredients except protein and agarose). Afterwards and if necessary the gel is dissected with microtools, a small incision opens the gel and liberates the crystal. An enzyme (agarase) can be used to digest the polysaccharide network. Otherwise, protein crystals extracted mechanically from gel are suitable for X-ray diffraction analyses at ambient [84,85] or cryogenic temperatures [86-88]. Crystals can be deprived of any matrix lipid after growth in cubic phases by enzymatic hydrolysis with Candida rugosa lipase [89,90]. Solubilisation with a detergent like octyl glucoside is an alternative in the latter case.

[19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36]

5.3. Perspectives The previous analysis of the literature, shows the great potential offered by the crystallisation procedures using gels. It is also clear that more studies are needed in order to study the usefulness of these technique towards the synthesis of crystals of other large organic and inorganic molecules such as MOF’s which have only been minimally explored. The evidence obtained so far in the preparation of different polymorphs offers the possibilities of perfecting ways of obtaining selectively one specific polymorph an aspect of interest both in the materials and pharmaceutical research areas. Organometallic compounds have not been crystallized using these techniques so there is room for research in this field as there is in the synthesis of inclusion complexes through diffusion and counter-diffusion techniques in gels.

[37] [38] [39] [40] [41] [42] [43] [44] [45] [46]

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