**2.1 Effects on crystal orientation**

During crystallization, the SAM constituent exposed at the SAM surface interacts with molecules present in the crystallizing solution. These interactions are specific and in turn, can cause the stabilization of specific crystal faces. When this happens, the crystals grow in a specific orientation, causing changes in crystal morphology. Many researchers have reported specific crystal orientation on SAMs, brought about by interactions of the crystallizing molecules with SAMs.

For example, Lee *et al*. employed SAMs of rigid thiols on gold surfaces in order to investigate the effects of interfacial molecular recognition on nucleation and growth of L-Alanine and DL-Valine crystals (Lee *et al.*, 2002). L-alanine crystallizes from water in the orthorhombic space group P21212 (a=6.025Å, b=12.324Å and c=5.783Å), with bipyramidal morphology, dominated by the {020}, {120}, {110} and {011} growth forms (Figure 1).

Fig. 1. L alanine crystal grown from aqueous solution. (Lee *et al.*, 2002)

In order to examine the effect of the SAMs on L-Alanine crystallization, L-Alanine was crystallized from solutions and also onto a variety of rigid SAMs of thiols: 4'-methyl-4 mercaptobiphenyl, 4'-hydroxy-4-mercaptobiphenyl and 4-(4-mercaptophenyl) pyridine. Powder X-ray diffraction patterns and interfacial angle measurements of the L-alanine crystals are shown in Figure 2. In methyl-terminated SAMs, L-alanine selectively nucleated on the (020) plane on the SAM surface while on the OH-terminated SAMs, Lalanine nucleated on an unobserved (200) side face. In both cases, the area of each crystal face was substantially larger than the other faces on the crystal. Crystallization of Lalanine on 4-(4-mercaptophenyl) pyridine resulted in the (011) face as the plane corresponding to nucleation. The preferential interaction was explained on the basis of hydrogen bonding between the pyridine surface and the amino and methyl groups protruding out of the (011) plane. Figure 2 reveals that L-alanine crystals nucleating on SAM surfaces crystallize in an orthorhombic space group with similar unit cell dimensions. However, the functionalized SAMs have an effect on the nucleating plane and ultimately, on L-alanine crystal growth.

Crystallization on Self Assembled Monolayers 43

Fig. 3. X-ray diffraction spectra of DL valine nucleated on functional SAMs compared with DL valine crystallized from aqueous solution (top) and crystallographic images and morphology of DL valine crystallized on (a,b) 4-(4-mercaptophenyl) pyridine SAMs, (c,d) 4'-hydroxy-4-mercaptobiphenyl SAMs and (e,f) 4'-methyl-4-mercaptobiphenyl SAMs –

An additional example for the effect of SAMs on crystallization can be demonstrated in the work of Aizenberg and co-workers, who studied the growth of oriented calcium carbonate crystals on X-terminated alkanethiol SAMs (HS(CH2)nX) (Aizenberg *et al.*, 1999). The SAMs were terminated with a variety of functional groups (X=CO2-, SO3-, PO32-, OH, N(CH3)3+,CH3) and two different metal support films were used – Ag and Au. The calcium carbonate crystals were characterized by a variety of techniques – optical microscopy, SEM with image analysis and X-ray diffraction. Their results showed that SAMs terminated in acidic functionalities were found to induce nucleation more effectively than control surfaces of bare metal films, whereas SAMs terminated in CH3 or slightly basic N(CH3)3 actually inhibited their nucleation. The orientation of the crystals was highly homogeneous for each surface and depended on the functional group exposed at the SAM surface. The authors achieved a very high level of control over the orientations of the crystals. They were able to selectively nucleate calcite from six crystallographic planes. This was the first time that calcite had been grown in such a variety of orientations in an artificially controlled system.

Polymorphism is the ability of a molecule to crystallize in more than one packing arrangement. Polymorphs (Chieng et al., 2011) may possess significantly different structural and physical properties and therefore polymorphism has broad practical implications in solid state chemistry, materials science and pharmacology. Many methods have been developed to control polymorphism including tailor made additives (Yokota et al., 2006), surfactants (Garti and Zour, 1997) and varying solvents and temperature (Weissbuch et al., 2005). Recently, self assembled monolayers have also been shown to control crystal

Hiremath *et al.* applied SAM templates in order to control the polymorphism of 1,3-bis(*m*nitrophenyl) urea (MNPU – Figure 4). (Hiremath *et al.*, 2005) Previously studied, MNPU was found to have at least three different polymorphs - α, β and γ. The polymorphs are visually

(bottom). (Lee *et al.*, 2002)

**2.2 Effects on polymorphism** 

polymorphism.

Fig. 2. X-ray diffraction spectra of L alanine nucleated on functional SAMs compared with L alanine crystallized from aqueous solution (left) and crystallographic images and morphology of L-alanine crystallized on (a,b) 4'-methyl-4-mercaptobiphenyl SAMs, (c,d) 4' hydroxy-4-mercaptobiphenyl SAMs and (e,f) 4-(4-mercaptophenyl) pyridine SAMs – (right). (Lee *et al.*, 2002)

DL-Valine crystallizes in the monoclinic space group P21/c with unit cell dimensions (a=5.21Å, b=22.10Å, c=5.41Å and β=109.2°). In aqueous solution, DL-valine crystallizes as hexagonal platelets dominated by a slow-growing (020) flat face with (100), (002) and (202) side faces. DL-valine was crystallized on 4'-methyl-4-mercaptobiphenyl, 4'-hydroxy-4 mercaptobiphenyl and 4-(4-mercaptophenyl) pyridine. On the pyridine and OH-terminated thiols, DL-valine crystals nucleated from the flat (020) plane, whereas in methyl terminated SAMs, the fast growing (100) face was the nucleating plane and the hexagonal platelet crystals were oriented perpendicular to the SAM surface (Figure 3). Again, the surface chemistry of the SAMs led to different interfacial interactions and thus oriented the crystallization of amino acids. In both cases, hydrogen bonding was responsible for the epitaxial crystallization of the amino acid on the monolayer substrates. Molecular modelling studies were also undertaken to examine the molecular recognition between the monolayer and the crystallographic planes and were in good agreement with the experimental results.

Fig. 2. X-ray diffraction spectra of L alanine nucleated on functional SAMs compared with L

morphology of L-alanine crystallized on (a,b) 4'-methyl-4-mercaptobiphenyl SAMs, (c,d) 4' hydroxy-4-mercaptobiphenyl SAMs and (e,f) 4-(4-mercaptophenyl) pyridine SAMs – (right).

DL-Valine crystallizes in the monoclinic space group P21/c with unit cell dimensions (a=5.21Å, b=22.10Å, c=5.41Å and β=109.2°). In aqueous solution, DL-valine crystallizes as hexagonal platelets dominated by a slow-growing (020) flat face with (100), (002) and (202) side faces. DL-valine was crystallized on 4'-methyl-4-mercaptobiphenyl, 4'-hydroxy-4 mercaptobiphenyl and 4-(4-mercaptophenyl) pyridine. On the pyridine and OH-terminated thiols, DL-valine crystals nucleated from the flat (020) plane, whereas in methyl terminated SAMs, the fast growing (100) face was the nucleating plane and the hexagonal platelet crystals were oriented perpendicular to the SAM surface (Figure 3). Again, the surface chemistry of the SAMs led to different interfacial interactions and thus oriented the crystallization of amino acids. In both cases, hydrogen bonding was responsible for the epitaxial crystallization of the amino acid on the monolayer substrates. Molecular modelling studies were also undertaken to examine the molecular recognition between the monolayer and the crystallographic planes and were in good agreement with the experimental results.

alanine crystallized from aqueous solution (left) and crystallographic images and

(Lee *et al.*, 2002)

Fig. 3. X-ray diffraction spectra of DL valine nucleated on functional SAMs compared with DL valine crystallized from aqueous solution (top) and crystallographic images and morphology of DL valine crystallized on (a,b) 4-(4-mercaptophenyl) pyridine SAMs, (c,d) 4'-hydroxy-4-mercaptobiphenyl SAMs and (e,f) 4'-methyl-4-mercaptobiphenyl SAMs – (bottom). (Lee *et al.*, 2002)

An additional example for the effect of SAMs on crystallization can be demonstrated in the work of Aizenberg and co-workers, who studied the growth of oriented calcium carbonate crystals on X-terminated alkanethiol SAMs (HS(CH2)nX) (Aizenberg *et al.*, 1999). The SAMs were terminated with a variety of functional groups (X=CO2-, SO3-, PO3 2-, OH, N(CH3)3+,CH3) and two different metal support films were used – Ag and Au. The calcium carbonate crystals were characterized by a variety of techniques – optical microscopy, SEM with image analysis and X-ray diffraction. Their results showed that SAMs terminated in acidic functionalities were found to induce nucleation more effectively than control surfaces of bare metal films, whereas SAMs terminated in CH3 or slightly basic N(CH3)3 actually inhibited their nucleation. The orientation of the crystals was highly homogeneous for each surface and depended on the functional group exposed at the SAM surface. The authors achieved a very high level of control over the orientations of the crystals. They were able to selectively nucleate calcite from six crystallographic planes. This was the first time that calcite had been grown in such a variety of orientations in an artificially controlled system.

#### **2.2 Effects on polymorphism**

Polymorphism is the ability of a molecule to crystallize in more than one packing arrangement. Polymorphs (Chieng et al., 2011) may possess significantly different structural and physical properties and therefore polymorphism has broad practical implications in solid state chemistry, materials science and pharmacology. Many methods have been developed to control polymorphism including tailor made additives (Yokota et al., 2006), surfactants (Garti and Zour, 1997) and varying solvents and temperature (Weissbuch et al., 2005). Recently, self assembled monolayers have also been shown to control crystal polymorphism.

Hiremath *et al.* applied SAM templates in order to control the polymorphism of 1,3-bis(*m*nitrophenyl) urea (MNPU – Figure 4). (Hiremath *et al.*, 2005) Previously studied, MNPU was found to have at least three different polymorphs - α, β and γ. The polymorphs are visually

Crystallization on Self Assembled Monolayers 45

α-polymorph is obtained exclusively. Dressler and Mastai demonstrated that the α-form can be obtained at room temperature, without the need for crystallization at low temperatures.

Table 1. Crystal of MNPU grown on SAMs 1-7 including the initial solution phase, solvent

Substrates for SAM preparation were prepared by vacuum deposition of thin gold films onto glass slides. The gold substrates were then immersed in 2mM L-AAPP ethanolic solutions overnight in order to receive the L-AAPP SAMs. L-AAPP was chosen as the molecule for self assembly since L-phenylalanine has already been shown to promote the stabilization of the α-form of L-glutamic acid in solution. In order to examine the effect of the SAMs on L-glutamic acid crystallization, a series of crystallization experiments were performed on the surfaces and in solutions. The crystals were then identified using various techniques (Figure 6 and Figure 7). The X-ray diffraction and Raman spectra reveal that the crystals grown on the SAMs are of the α-form and those from solution are β-L-glutamic acid. From X-ray diffraction, It is also evident that L-glutamic acid crystallizes on the surfaces with preferential crystal growth along the <111> crystal plane. It is also clear from the SEM images that the polymorphs possess very different crystal habits. Crystals grown on the SAM surfaces reveal well-ordered large prismatic structures typical of α-L-glutamic acid, whereas crystals from solution expose plate like structures characteristic of the β-form. These results verify the stabilization of the α-polymorph of L-glutamic acid on the L-phenylalanine terminated SAM. The stabilization is explained in the following manner: β-L-glutamic acid emerges from the (011) plane of α-L-glutamic acid. L-Phenylalanine attaches to the (011) plane of the α-crystals, thereby inhibiting and preventing the α to β

and temperature crystallization parameters. (Hiremath *et al.*, 2005)

Fig. 5. L-AAPP (Dressler and Mastai, 2007)

transformation.

distinguishable, but often crystallize concomitantly from aqueous ethanol solutions. The crystal structures of the α and β polymorphs were first reported by Etter et al (Etter *et. al*, 1988, 1990, as cited in Hiremath *et al.*, 2005). Bernstein *et al.* (Bernstein *et al.*, 2005, as cited in Hiremath *et al.*, 2005) obtained the crystal structure of the γ phase and also discovered a previously unknown anhydrous δ phase.

Fig. 4. MNPU (right) and biphenyl thiol molecules used in the biphenyl thiol SAMs (1-7) – (left). (Hiremath *et al.*, 2005)

Initially, Hiremath *et al.* obtained the pure polymorph phases by varying solvents and temperature. The α-MNPU was obtained from crystallization in ethanol at 60°C, β-MNPU was obtained from crystallization in ethyl acetate at room temperature. The γ form was obtained by re-crystallization of the α or β forms in 95% ethanol. Then, MNPU was crystallized on a variety of biphenyl thiol SAMs (Figure 4) and two types of control surfaces: bare gold substrates and 1-pentanethiol, 1-octanethiol and 1-dodecanethiol SAMs. The biphenyl thiols were chosen for two reasons – their close packing affords a more perpendicular molecular alignment at the surface (smaller tilt angles) and the slightly larger size of the 2D lattice dimensions makes them potentially more compatible with typical unit cell dimensions. The crystallizations were conducted in saturated ethanol or ethyl acetate solutions of MNPU at room temperature or 60°C. The results obtained from the crystallizations are summarized in Table 1. Crystals of MNPU only appeared on SAMs 1-3. No crystallization of MNPU was observed on SAMs 4-7 or on the control surfaces. SAMs of 1, 2 and 3 were each found to serve as selective nucleating templates for a single MNPU phase. Crystals of α-MNPU formed on monolayers of 1, β-MNPU grew selectively on 2 and γ-MNPU grew on 3. The crystallizations were repeated numerous times and the results were always consistent. The resultant crystal form obtained was independent of the solvent or temperature conditions explored. In addition, the crystals grown on SAMs 1-3 all adopted preferred orientations with respect to the SAM templates, illustrating the importance of the interface in the nucleation process. The observed orientations were rationalized on the basis of two-dimensional lattice matching, complementary functional group interactions and dipole moments across the SAM/crystal interface.

The work of Dressler and Mastai provides an additional example of polymorphism control by SAMs. Dressler and Mastai crystallized the metastable α-L-Glutamic acid on L-Phenylalanine SAMs (L-AAPP, Figure 5) (Dressler and Mastai, 2007). L-Glutamic acid is known to form two polymorphs – the metastable α-form and the stable β-form. It was previously shown that the stable β-form emerges from the metastable α-crystals through a kinetic process. It was also shown that the α-polymorph converts rapidly to the β-form at 45°C, but at ca. 15°C, the conversion process is very slow and almost nonexistent, so that the

distinguishable, but often crystallize concomitantly from aqueous ethanol solutions. The crystal structures of the α and β polymorphs were first reported by Etter et al (Etter *et. al*, 1988, 1990, as cited in Hiremath *et al.*, 2005). Bernstein *et al.* (Bernstein *et al.*, 2005, as cited in Hiremath *et al.*, 2005) obtained the crystal structure of the γ phase and also discovered a

Fig. 4. MNPU (right) and biphenyl thiol molecules used in the biphenyl thiol SAMs

Initially, Hiremath *et al.* obtained the pure polymorph phases by varying solvents and temperature. The α-MNPU was obtained from crystallization in ethanol at 60°C, β-MNPU was obtained from crystallization in ethyl acetate at room temperature. The γ form was obtained by re-crystallization of the α or β forms in 95% ethanol. Then, MNPU was crystallized on a variety of biphenyl thiol SAMs (Figure 4) and two types of control surfaces: bare gold substrates and 1-pentanethiol, 1-octanethiol and 1-dodecanethiol SAMs. The biphenyl thiols were chosen for two reasons – their close packing affords a more perpendicular molecular alignment at the surface (smaller tilt angles) and the slightly larger size of the 2D lattice dimensions makes them potentially more compatible with typical unit cell dimensions. The crystallizations were conducted in saturated ethanol or ethyl acetate solutions of MNPU at room temperature or 60°C. The results obtained from the crystallizations are summarized in Table 1. Crystals of MNPU only appeared on SAMs 1-3. No crystallization of MNPU was observed on SAMs 4-7 or on the control surfaces. SAMs of 1, 2 and 3 were each found to serve as selective nucleating templates for a single MNPU phase. Crystals of α-MNPU formed on monolayers of 1, β-MNPU grew selectively on 2 and γ-MNPU grew on 3. The crystallizations were repeated numerous times and the results were always consistent. The resultant crystal form obtained was independent of the solvent or temperature conditions explored. In addition, the crystals grown on SAMs 1-3 all adopted preferred orientations with respect to the SAM templates, illustrating the importance of the interface in the nucleation process. The observed orientations were rationalized on the basis of two-dimensional lattice matching, complementary functional group interactions and

The work of Dressler and Mastai provides an additional example of polymorphism control by SAMs. Dressler and Mastai crystallized the metastable α-L-Glutamic acid on L-Phenylalanine SAMs (L-AAPP, Figure 5) (Dressler and Mastai, 2007). L-Glutamic acid is known to form two polymorphs – the metastable α-form and the stable β-form. It was previously shown that the stable β-form emerges from the metastable α-crystals through a kinetic process. It was also shown that the α-polymorph converts rapidly to the β-form at 45°C, but at ca. 15°C, the conversion process is very slow and almost nonexistent, so that the

previously unknown anhydrous δ phase.

(1-7) – (left). (Hiremath *et al.*, 2005)

dipole moments across the SAM/crystal interface.

α-polymorph is obtained exclusively. Dressler and Mastai demonstrated that the α-form can be obtained at room temperature, without the need for crystallization at low temperatures.


Table 1. Crystal of MNPU grown on SAMs 1-7 including the initial solution phase, solvent and temperature crystallization parameters. (Hiremath *et al.*, 2005)

Fig. 5. L-AAPP (Dressler and Mastai, 2007)

Substrates for SAM preparation were prepared by vacuum deposition of thin gold films onto glass slides. The gold substrates were then immersed in 2mM L-AAPP ethanolic solutions overnight in order to receive the L-AAPP SAMs. L-AAPP was chosen as the molecule for self assembly since L-phenylalanine has already been shown to promote the stabilization of the α-form of L-glutamic acid in solution. In order to examine the effect of the SAMs on L-glutamic acid crystallization, a series of crystallization experiments were performed on the surfaces and in solutions. The crystals were then identified using various techniques (Figure 6 and Figure 7). The X-ray diffraction and Raman spectra reveal that the crystals grown on the SAMs are of the α-form and those from solution are β-L-glutamic acid. From X-ray diffraction, It is also evident that L-glutamic acid crystallizes on the surfaces with preferential crystal growth along the <111> crystal plane. It is also clear from the SEM images that the polymorphs possess very different crystal habits. Crystals grown on the SAM surfaces reveal well-ordered large prismatic structures typical of α-L-glutamic acid, whereas crystals from solution expose plate like structures characteristic of the β-form. These results verify the stabilization of the α-polymorph of L-glutamic acid on the L-phenylalanine terminated SAM. The stabilization is explained in the following manner: β-L-glutamic acid emerges from the (011) plane of α-L-glutamic acid. L-Phenylalanine attaches to the (011) plane of the α-crystals, thereby inhibiting and preventing the α to β transformation.

Crystallization on Self Assembled Monolayers 47

SAMs were then formed on the gold surfaces by immersing the gold substrates in 0.1M solutions of S-LMHA in ethanol overnight. In order to examine the effect of the chiral SAMs on the DL-glutamic acid crystallization, DL-glutamic acid was crystallized from aqueous solution onto the S-LMHA chiral SAMs. DL-glutamic acid was also crystallized in solution, under the same conditions. The crystals from solution and those grown on the chiral SAMs were examined using X-ray diffraction (XRD), scanning electron microscopy (SEM), micro-Raman and differential scanning calorimetry (DSC). It should be mentioned that

The X-ray diffraction spectrum of crystals from solution is shown in Figure 9 and corresponds to monohydrated DL-glutamic acid, namely the racemic compound, as reported in the literature. The monohydrate of DL-glutamic acid crystallizes in an orthorhombic unit cell (space group Pbca) with the following parameters a=9.08, b = 15.40, c = 10.61 (A) and α = β = λ = 90°. On the other hand, the X-ray diffraction patterns of crystals grown on S-LMHA that were taken directly on the chiral surface show a typical Xray diffraction spectrum of the conglomerate, anhydrous DL-glutamic acid. The X-ray diffraction spectrum of conglomerate DL-glutamic acid corresponds to that reported in the literature with unit cell parameters (in A): a=5.16, b=17.30, c=6.95, α = β = λ = 90°

The anhydrous and monohydrated forms of DL-glutamic acid have different crystal habits and therefore their formation was also studied using SEM. Figure 9 displays SEM images of crystals collected from solution and crystals grown on the S-LMHA SAMs. The crystals

Fig. 9. X-Ray diffraction spectra of (A) DL-glutamic acid crystals grown on S-LMHA chiral SAMs (anhydrous DL-glutamic acid) and (B) crystals grown in solution (DL-glutamic acid monohydrate) and SEM images of (C) crystals grown in solution (scale bar = 1 mm) (DL-glutamic acid monohydrate) and (D) crystals grown on S-LMHA chiral SAMs (scale bar = 500 mm) (anhydrous DL-glutamic acid). (Ejgenberg and Mastai, 2011)

crystallization or crystal growth did not occur on the bare gold surfaces.

(orthorhombic unit cell, space group P212121).

Fig. 6. X-ray diffraction of L glutamic acid crystals (A) grown on L-AAPP SAMs, (B) grown on Au and (C) grown from solution – (left) and Raman spectroscopy of L glutamic acid crystals (A) grown on L-AAPP SAMs (α-polymorph) and (B) grown from solution (β-polymorph). (Dressler and Mastai, 2007)

Fig. 7. Scanning electron microscopy images of L glutamic acid crystals (scale bar=200μm): (A) crystal morphology of crystals grown on the surface of L-AAPP SAMs and (B) crystal morphology of crystals grown from solution. (Dressler and Mastai, 2007)

Recently, Ejgenberg and Mastai demonstrated that self assembled monolayers based on S-leucine (Figure 8) can be used for polymorphism control of DL glutamic acid (Ejgenberg and Mastai, 2011). It is known that DL-glutamic acid can crystallize in two forms: the conglomerate form, known as anhydrous DL glutamic acid and the racemic compound, known as DL glutamic acid monohydrate. Ejgenberg and Mastai were able to afford anhydrous DL glutamic acid under conditions where the monohydrated DL glutamic acid is thermodynamically more stable.

Fig. 8. S-leucine methyl ester covalently attached to 6-mercaptohexanoic acid (S-LMHA). Red atoms = oxygen, blue =nitrogen, gray = carbon, yellow = sulfur, white = hydrogen. (Ejgenberg and Mastai, 2011)

The chiral SAMs were prepared on ultra-flat gold surfaces. Gold films of 50 nm thickness were deposited on mica substrates using a high vacuum sputtering technique. The chiral

Fig. 6. X-ray diffraction of L glutamic acid crystals (A) grown on L-AAPP SAMs, (B) grown on Au and (C) grown from solution – (left) and Raman spectroscopy of L glutamic acid crystals (A) grown on L-AAPP SAMs (α-polymorph) and (B) grown from solution

Fig. 7. Scanning electron microscopy images of L glutamic acid crystals (scale bar=200μm): (A) crystal morphology of crystals grown on the surface of L-AAPP SAMs and (B) crystal

Recently, Ejgenberg and Mastai demonstrated that self assembled monolayers based on S-leucine (Figure 8) can be used for polymorphism control of DL glutamic acid (Ejgenberg and Mastai, 2011). It is known that DL-glutamic acid can crystallize in two forms: the conglomerate form, known as anhydrous DL glutamic acid and the racemic compound, known as DL glutamic acid monohydrate. Ejgenberg and Mastai were able to afford anhydrous DL glutamic acid under conditions where the monohydrated DL glutamic acid is thermodynamically more stable.

Fig. 8. S-leucine methyl ester covalently attached to 6-mercaptohexanoic acid (S-LMHA). Red atoms = oxygen, blue =nitrogen, gray = carbon, yellow = sulfur, white = hydrogen.

The chiral SAMs were prepared on ultra-flat gold surfaces. Gold films of 50 nm thickness were deposited on mica substrates using a high vacuum sputtering technique. The chiral

morphology of crystals grown from solution. (Dressler and Mastai, 2007)

(β-polymorph). (Dressler and Mastai, 2007)

(Ejgenberg and Mastai, 2011)

SAMs were then formed on the gold surfaces by immersing the gold substrates in 0.1M solutions of S-LMHA in ethanol overnight. In order to examine the effect of the chiral SAMs on the DL-glutamic acid crystallization, DL-glutamic acid was crystallized from aqueous solution onto the S-LMHA chiral SAMs. DL-glutamic acid was also crystallized in solution, under the same conditions. The crystals from solution and those grown on the chiral SAMs were examined using X-ray diffraction (XRD), scanning electron microscopy (SEM), micro-Raman and differential scanning calorimetry (DSC). It should be mentioned that crystallization or crystal growth did not occur on the bare gold surfaces.

The X-ray diffraction spectrum of crystals from solution is shown in Figure 9 and corresponds to monohydrated DL-glutamic acid, namely the racemic compound, as reported in the literature. The monohydrate of DL-glutamic acid crystallizes in an orthorhombic unit cell (space group Pbca) with the following parameters a=9.08, b = 15.40, c = 10.61 (A) and α = β = λ = 90°. On the other hand, the X-ray diffraction patterns of crystals grown on S-LMHA that were taken directly on the chiral surface show a typical Xray diffraction spectrum of the conglomerate, anhydrous DL-glutamic acid. The X-ray diffraction spectrum of conglomerate DL-glutamic acid corresponds to that reported in the literature with unit cell parameters (in A): a=5.16, b=17.30, c=6.95, α = β = λ = 90° (orthorhombic unit cell, space group P212121).

The anhydrous and monohydrated forms of DL-glutamic acid have different crystal habits and therefore their formation was also studied using SEM. Figure 9 displays SEM images of crystals collected from solution and crystals grown on the S-LMHA SAMs. The crystals

Fig. 9. X-Ray diffraction spectra of (A) DL-glutamic acid crystals grown on S-LMHA chiral SAMs (anhydrous DL-glutamic acid) and (B) crystals grown in solution (DL-glutamic acid monohydrate) and SEM images of (C) crystals grown in solution (scale bar = 1 mm) (DL-glutamic acid monohydrate) and (D) crystals grown on S-LMHA chiral SAMs (scale bar = 500 mm) (anhydrous DL-glutamic acid). (Ejgenberg and Mastai, 2011)

Crystallization on Self Assembled Monolayers 49

Fig. 11. DSC thermograms of (A) DL-glutamic acid crystals grown in solution (DL-glutamic acid monohydrate) and (B) crystals grown on S-LMHA chiral SAMs (anhydrous DL-

Patterned SAMs are composed of two or more SAMs which are deliberately distributed on specific areas on the surface. They can be fabricated in different ways including microcontact printing and dip-pen nanolithography. Patterned SAMs have been used to attract particular nanostructures like ribbons or wires to particular areas on the surface. They have also been used in the functionalization of biosensors in which the localized SAMs have an affinity for specific cells and proteins. Crystallization on patterned SAMs where the crystals

In a fascinating study, Aizenberg *et al.* demonstrated that crystallization could be restricted to well-defined regions on micro-patterned SAMs (Aizenberg *et al.*, 1999). The substrates were used in order to control the crystallization of calcite. Calcite is a stable polymorph of calcium carbonate, with space group R3c, α = 4.99Å and c = 17.06Å. Calcite has been

Self assembled monolayers having areas of different nucleating activity were patterned on metal substrates. The self assembled monolayers were patterned by microcontact printing with an elastomeric stamp that had a relief structure consisting of a square array of raised circles: as inks, 10mM solutions of HS(CH2)nX (X=CO2H) in ethanol were used. The surface was then washed with a 10mM solution of HS(CH2)15CH3 in ethanol to passivate the areas that had not contacted the stamp. The patterned substrates were supported upside down in the crystallizing solutions to ensure that only particles grown on the SAM would be bound to the surface.

Figure 12 shows a low magnification image of calcite crystals formed on one of the patterned SAMs. It is clear that crystallization is restricted to well defined, CO2 – terminated regions and does not occur on the CH3 – terminated areas. Interestingly, by adjusting parameters such as the density and size features on the stamp, Aizenberg *et al.* could control important crystallization characteristics, including the location and density of nucleating regions on the surface, the number of crystals nucleated in each region and the crystallographic orientation of the crystals (Figure 13). The following mechanism was suggested: the rate of nucleation on SAMs

glutamic acid). (Ejgenberg and Mastai, 2011)

grow in localized areas has also been demonstrated.

extensively studied and its crystallization is relatively easy to perform.

**2.3 Crystallization on patterned SAMs** 

grown from solution reveal typical needle-like morphology, characteristic of monohydrated DL-glutamic acid. The crystals are fairly large and non-uniform in size (1–4 mm long and 0.2–0.5 mm wide). However, crystals grown on the S-LMHA SAMs expose plate like morphology characteristic of anhydrous DL-glutamic acid. They are uniform in size with approximately 250 X 250 µm2 and are much smaller than the crystals from solution.

In order to further characterize the conglomerate and racemic compound DL-glutamic acid crystals, micro-Raman measurements were carried out. The Raman spectrum of DL-glutamic acid crystallized on the S-LMHA surface is shown in Figure 10 with main peaks at 867, 1295, 1339 and 1405 cm\_1. The Raman spectrum of DL-glutamic acid crystallized from solution (Figure 10) displays main peaks at 855, 919 and 1314 cm\_1. These spectra correspond to the Raman spectra of conglomerate and racemic glutamic acid as reported in the literature. The Raman measurements of DL-glutamic acid crystallized on the chiral surface match those of the conglomerate form of DL-glutamic acid.

Fig. 10. Raman spectra of (A) DL-glutamic acid crystals grown on S-LMHA chiral SAMs (anhydrous DL-glutamic acid) and (B) crystals grown in solution (DL-glutamic acid monohydrate). (Ejgenberg and Mastai, 2011)

Further evidence for the formation of the conglomerate DL-glutamic acid crystals on the chiral SAM surfaces was obtained from differential scanning calorimetry (DSC) (Figure 11). The DSC thermogram of the DL-glutamic crystals grown on the S-LMHA SAMs exhibits a sharp melting point at 185.4 1C. The DSC thermogram of the crystals from solution shows two endothermic peaks at 112.7 1C and 184.7 1C. These results can be explained in the following manner. The crystals grown on the chiral SAMs are anhydrous DL-glutamic acid and therefore exhibit one peak, while those from solution are monohydrated DL-glutamic acid and therefore contain two peaks: the first endotherm is due to the loss of water molecules and the second sharp endotherm is due to the melting of anhydrous DL-glutamic acid.

In conclusion, S-LMHA SAMs were used to stabilize the conglomerate form of DL-glutamic acid crystals. The results offer a powerful tool in the development of processes for controlling chiral polymorphic systems and can further develop into a novel method for chiral resolution by crystallization.

grown from solution reveal typical needle-like morphology, characteristic of monohydrated DL-glutamic acid. The crystals are fairly large and non-uniform in size (1–4 mm long and 0.2–0.5 mm wide). However, crystals grown on the S-LMHA SAMs expose plate like morphology characteristic of anhydrous DL-glutamic acid. They are uniform in size with approximately 250 X 250 µm2 and are much smaller than the crystals

In order to further characterize the conglomerate and racemic compound DL-glutamic acid crystals, micro-Raman measurements were carried out. The Raman spectrum of DL-glutamic acid crystallized on the S-LMHA surface is shown in Figure 10 with main peaks at 867, 1295, 1339 and 1405 cm\_1. The Raman spectrum of DL-glutamic acid crystallized from solution (Figure 10) displays main peaks at 855, 919 and 1314 cm\_1. These spectra correspond to the Raman spectra of conglomerate and racemic glutamic acid as reported in the literature. The Raman measurements of DL-glutamic acid crystallized on the

Fig. 10. Raman spectra of (A) DL-glutamic acid crystals grown on S-LMHA chiral SAMs (anhydrous DL-glutamic acid) and (B) crystals grown in solution (DL-glutamic acid

Further evidence for the formation of the conglomerate DL-glutamic acid crystals on the chiral SAM surfaces was obtained from differential scanning calorimetry (DSC) (Figure 11). The DSC thermogram of the DL-glutamic crystals grown on the S-LMHA SAMs exhibits a sharp melting point at 185.4 1C. The DSC thermogram of the crystals from solution shows two endothermic peaks at 112.7 1C and 184.7 1C. These results can be explained in the following manner. The crystals grown on the chiral SAMs are anhydrous DL-glutamic acid and therefore exhibit one peak, while those from solution are monohydrated DL-glutamic acid and therefore contain two peaks: the first endotherm is due to the loss of water molecules and the second sharp endotherm is due to the melting of anhydrous DL-glutamic

In conclusion, S-LMHA SAMs were used to stabilize the conglomerate form of DL-glutamic acid crystals. The results offer a powerful tool in the development of processes for controlling chiral polymorphic systems and can further develop into a novel method for

monohydrate). (Ejgenberg and Mastai, 2011)

chiral resolution by crystallization.

chiral surface match those of the conglomerate form of DL-glutamic acid.

from solution.

acid.

Fig. 11. DSC thermograms of (A) DL-glutamic acid crystals grown in solution (DL-glutamic acid monohydrate) and (B) crystals grown on S-LMHA chiral SAMs (anhydrous DLglutamic acid). (Ejgenberg and Mastai, 2011)

#### **2.3 Crystallization on patterned SAMs**

Patterned SAMs are composed of two or more SAMs which are deliberately distributed on specific areas on the surface. They can be fabricated in different ways including microcontact printing and dip-pen nanolithography. Patterned SAMs have been used to attract particular nanostructures like ribbons or wires to particular areas on the surface. They have also been used in the functionalization of biosensors in which the localized SAMs have an affinity for specific cells and proteins. Crystallization on patterned SAMs where the crystals grow in localized areas has also been demonstrated.

In a fascinating study, Aizenberg *et al.* demonstrated that crystallization could be restricted to well-defined regions on micro-patterned SAMs (Aizenberg *et al.*, 1999). The substrates were used in order to control the crystallization of calcite. Calcite is a stable polymorph of calcium carbonate, with space group R3c, α = 4.99Å and c = 17.06Å. Calcite has been extensively studied and its crystallization is relatively easy to perform.

Self assembled monolayers having areas of different nucleating activity were patterned on metal substrates. The self assembled monolayers were patterned by microcontact printing with an elastomeric stamp that had a relief structure consisting of a square array of raised circles: as inks, 10mM solutions of HS(CH2)nX (X=CO2H) in ethanol were used. The surface was then washed with a 10mM solution of HS(CH2)15CH3 in ethanol to passivate the areas that had not contacted the stamp. The patterned substrates were supported upside down in the crystallizing solutions to ensure that only particles grown on the SAM would be bound to the surface.

Figure 12 shows a low magnification image of calcite crystals formed on one of the patterned SAMs. It is clear that crystallization is restricted to well defined, CO2 – terminated regions and does not occur on the CH3 – terminated areas. Interestingly, by adjusting parameters such as the density and size features on the stamp, Aizenberg *et al.* could control important crystallization characteristics, including the location and density of nucleating regions on the surface, the number of crystals nucleated in each region and the crystallographic orientation of the crystals (Figure 13). The following mechanism was suggested: the rate of nucleation on SAMs

Crystallization on Self Assembled Monolayers 51

The crystallization experiments were conducted by immersing and withdrawing the patterned substrates from aqueous glycine solutions of three different pHs (neutral, acidic and basic) and concentrations. Solution droplets were created on the patterned surface and each droplet served as an independent crystallization trial. More than 2000 islands per substrate were analyzed to achieve the statistical accuracy of the polymorph distribution of

Table 2. Polymorph distribution of glycine for different pHs and solution concentrations.

pH of solution for acidic/basic solutions during evaporation.

Table 3. The polymorph outcome of glycine for different pHs and island sizes.

In similar studies, Lee *et al.* reported that the size of the glycine crystals is controlled by the dimensions of the metallic gold islands or the concentration of the solution (Lee, A.Y. *et al.*, 2005). Moreover, it was observed that the high energy unstable β-form of glycine crystallizes

The polymorph distribution of glycine crystals formed on the patterned substrates with respect to the solution concentration and pH are summarized in Table 2. For the neutral aqueous glycine solution, the α-form was preferred at all concentrations, but its frequency significantly decreased at acidic and basic solutions. Consequentially, the percentage of γglycine increased in the acidic and basic solutions, without any large difference in the polymorph distribution of the β-polymorph. The increased percentage of the γ-polymorph in acidic and basic solutions was rationalized in the following way: the acidic and basic glycine solutions contain charged glycine species which reduce the possibility of the formation of α-glycine. Table 3 summarizes the polymorph outcome of glycine for different pHs and island sizes. As shown in previous reports (Lee, A.Y. *et al.*, 2005, 2006), metastable forms (β-glycine) were consistently more frequently observed with smaller islands. However, using acidic and basic glycine solutions, the frequency of the thermodynamically most stable γ-glycine increased with decreasing island size. This can be attributed to the increased proportion of the charged glycine species caused by the decrease/increase in the

crystals formed on the substrate.

(Lee, I.S. *et al.*, 2008)

(Lee, I.S. *et al.*, 2008)

terminated in polar groups is faster than on methyl-terminated SAMs. As soon as crystal growth begins in a polar region, mass transport to the growing crystals depletes calcium and carbonate ions over the local methyl-terminated region to the point of under saturation.

Fig. 12. Scanning electron micrograph of a patterned surface – calcite crystals appear on circles of HS(CH2)15CO2H in a background of HS(CH2)15CH3. (Aizenberg *et al.*, 1999)

Fig. 13. Ordered two dimensional arrays of calcite crystals. (Aizenberg *et al.*, 1999)

Recently, Lee *et al.* investigated the concomitant crystallization of glycine polymorphs on patterned self assembled monolayers (Lee, I.S. *et al.*, 2008). Six distinct polymorphic forms of glycine are known in the literature; three of them – α, β and γ - are formed at ambient environment. At room temperature, γ-glycine is the thermodynamically most stable form. However, in neutral aqueous solution, the α-form is normally obtained. The γ-form is typically obtained from acidic and basic solutions. β-glycine is the least stable of the three forms. It can be obtained from ethanol-water mixtures, but readily converts to α-glycine in the presence of water or upon heating.

Patterned subtrates were designed in the following manner: metallic gold islands were formed by evaporating titanium followed by gold through brass meshes onto glass substrates. The dimensions and patterns of the islands depended on the size and shape of the holes on the mesh. Square shaped islands in a variety of sizes were prepared. 4- Mercaptobenzoic acid (4-MBA) was self assembled onto the gold surface while octadecyltrichlorosilane (OTS) was applied to the exposed glass surface.

terminated in polar groups is faster than on methyl-terminated SAMs. As soon as crystal growth begins in a polar region, mass transport to the growing crystals depletes calcium and

carbonate ions over the local methyl-terminated region to the point of under saturation.

Fig. 12. Scanning electron micrograph of a patterned surface – calcite crystals appear on circles of HS(CH2)15CO2H in a background of HS(CH2)15CH3. (Aizenberg *et al.*, 1999)

Fig. 13. Ordered two dimensional arrays of calcite crystals. (Aizenberg *et al.*, 1999)

the presence of water or upon heating.

Recently, Lee *et al.* investigated the concomitant crystallization of glycine polymorphs on patterned self assembled monolayers (Lee, I.S. *et al.*, 2008). Six distinct polymorphic forms of glycine are known in the literature; three of them – α, β and γ - are formed at ambient environment. At room temperature, γ-glycine is the thermodynamically most stable form. However, in neutral aqueous solution, the α-form is normally obtained. The γ-form is typically obtained from acidic and basic solutions. β-glycine is the least stable of the three forms. It can be obtained from ethanol-water mixtures, but readily converts to α-glycine in

Patterned subtrates were designed in the following manner: metallic gold islands were formed by evaporating titanium followed by gold through brass meshes onto glass substrates. The dimensions and patterns of the islands depended on the size and shape of the holes on the mesh. Square shaped islands in a variety of sizes were prepared. 4- Mercaptobenzoic acid (4-MBA) was self assembled onto the gold surface while

octadecyltrichlorosilane (OTS) was applied to the exposed glass surface.

The crystallization experiments were conducted by immersing and withdrawing the patterned substrates from aqueous glycine solutions of three different pHs (neutral, acidic and basic) and concentrations. Solution droplets were created on the patterned surface and each droplet served as an independent crystallization trial. More than 2000 islands per substrate were analyzed to achieve the statistical accuracy of the polymorph distribution of crystals formed on the substrate.


Table 2. Polymorph distribution of glycine for different pHs and solution concentrations. (Lee, I.S. *et al.*, 2008)

The polymorph distribution of glycine crystals formed on the patterned substrates with respect to the solution concentration and pH are summarized in Table 2. For the neutral aqueous glycine solution, the α-form was preferred at all concentrations, but its frequency significantly decreased at acidic and basic solutions. Consequentially, the percentage of γglycine increased in the acidic and basic solutions, without any large difference in the polymorph distribution of the β-polymorph. The increased percentage of the γ-polymorph in acidic and basic solutions was rationalized in the following way: the acidic and basic glycine solutions contain charged glycine species which reduce the possibility of the formation of α-glycine. Table 3 summarizes the polymorph outcome of glycine for different pHs and island sizes. As shown in previous reports (Lee, A.Y. *et al.*, 2005, 2006), metastable forms (β-glycine) were consistently more frequently observed with smaller islands. However, using acidic and basic glycine solutions, the frequency of the thermodynamically most stable γ-glycine increased with decreasing island size. This can be attributed to the increased proportion of the charged glycine species caused by the decrease/increase in the pH of solution for acidic/basic solutions during evaporation.


Table 3. The polymorph outcome of glycine for different pHs and island sizes. (Lee, I.S. *et al.*, 2008)

In similar studies, Lee *et al.* reported that the size of the glycine crystals is controlled by the dimensions of the metallic gold islands or the concentration of the solution (Lee, A.Y. *et al.*, 2005). Moreover, it was observed that the high energy unstable β-form of glycine crystallizes

Crystallization on Self Assembled Monolayers 53

In order to study the interactions between the chiral crystals and the chiral SAMs, pure enantiomers of glutamic acid were first crystallized on the L and D cysteine films. Interestingly, enantiomers of glutamic acid with identical chirality to that of the cysteine SAMs did not grow on the SAMs in a specific orientation and were unchanged from crystals grown in solution (Table 4). On the other hand, enantiomers with chirality opposite to that of the cysteine films grew in a preffered orientation. In either case, morphological differences between crystals grown on the SAMs and those grown in solution were not very significant.

Table 4. Crystal habits and crystal orientation of DL, D and L glutamic acid crystallized on

The chiral cysteine SAMs were then utilized for the enantioselective crystallization of glutamic acid. For this purpose, L and D cysteine SAMs were immersed in supersaturated solutions of DL-glutamic acid for ca. 2 hours. The crystals were then characterized using various techniques. It is important to note here that glutamic acid grows as a monohydrate crystal from water and therefore has a different structure from its enantiomers which were discussed previously. Figure 14 presents SEM images of glutamic acid monohydrate crystals grown on L and D cysteine surfaces and grown from solution. Crystals grown from solution have a needle-like morphology with an average size of 300 μm whereas crystals grown on the cysteine surfaces grow in well ordered arrays of rectangular/plate like shaped crystals with a typical size of 10-30 μm. X-ray diffraction measurements of the crystals showed

Fig. 14. SEM images featuring the crystal morphology of rac-glutamic acid crystallized (A) from solution, (B) on a chiral D-cysteine surface and (C) on a chiral L-cysteine surface.

The enantiomeric excess of the crystals grown on the chiral SAMs was investigated using polarimetry. The crystals were gently removed from the chiral films, dissolved in 5N HCl

cysteine films and in pure solution. (Dressler and Mastai, 2007)

preffered orientation of the glutamic acid crystals (Table 4).

(Dressler and Mastai, 2007)

on small metallic islands, in contrast to large islands, where the α-form is favoured. The increased frequency of the high energy form (β-glycine) with decreasing feature sizes is a result of the high supersaturation that is generated from fast solvent evaporation. (Lee, A.Y. et al., 2006)
