脯氨酸雷射捕陷結晶化於溶液中之研究

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國

立

交

通

大

學

應用化學系碩士班

碩

士論文

脯氨酸雷射捕陷結晶化於溶液中之研究

Laser Trapping Crystallization of L-proline in Solution

研究生：黃重維 (Chong-Wei Huang)

指導教授：三浦篤志博士 (Dr. Atsushi Miura)

中華民國一百年七月

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脯氨酸雷射捕陷結晶化於溶液中之研究

Laser trapping crystallization of L-proline in solution

研究生：黃重維 Student：Chong-Wei Huang

指導教授：三浦篤志博士 Advisor：Dr. Atsushi Miura

國立交通大學

應用化學系碩士班

碩士論文

A Thesis

Submitted to M. S. Program

Department of Applied Chemistry

National Chiao Tung University

in Partial Fulfillment of the Requirements

for the Degree of

Master

in

Applied Chemistry

June 2011

Hsinchu, Taiwan, Republic of China

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Laser Trapping Crystallization of L-proline in Solution

Student：Chong-Wei Huang Advisor：Dr. Atsushi Miura

M. S. Program, Department of Applied Chemistry

National Chiao Tung University

Abstract

Photon pressure induced well-ordered molecular assembly formation technique, which has developed in our group and named as “laser trapping crystallization”, is quite unique and high potential method since its spatiotemporal controllability of crystallization. Meanwhile crystallization under laser trapping is not always successful and its mechanism has not been clarified.

In this work, we studied laser trapping crystallization of a natural amino acid L-proline to clarify its crystallization dynamics and mechanism under laser trapping condition. Laser trapping crystallization of L-proline in different solvents was examined by focusing a trapping laser to the air/solution interface. We observed obvious difference of crystallization behavior depending on the solvent.

We found that the laser trapping crystallization of proline in deuterated water (D2O) is

difficult. Meanwhile in deuterated ethanol (EtOD), we succeeded the laser trapping crystallization. Crystallization of proline in EtOD is very similar to previously reported

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laser trapping crystallization of glycine. Microscopic characterization of formed crystals indicates we successfully formed proline crystal by laser trapping.

In addition to crystallization, we observed locally induced liquid-liquid phase separation of proline in EtOD prior to the crystal formation, namely dense liquid droplet formation, as well as glycine in D2O solution. Detailed observation of droplet formation dynamics

enabled us to understand droplet formation dynamics and propose its mechanism.

Observed results indicate the dense droplet formation is indispensable process for laser trapping crystallization of proline in EtOD and laser trapping assembly formation, i.e. both crystallization and droplet formation, is dominated by complex contribution of various factors such as solvent characteristics, interactions between solvent and solute or solutes, and laser-induced local environment change around focal spot.

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脯氨酸雷射捕陷結晶化於溶液中之研究

研究生：黃重維指導教授：三浦篤志博士國立交通大學應用化學系碩士班

中文摘要

雷射捕捉促使分子聚集化最近引起大家的重視，特別是它所引起更進一步的分子聚集化現象:結晶化，我們稱之為“雷射捕陷結晶化”。第一次成功示範出雷射捕陷結晶化，是在過飽和的甘氨酸重水溶液中，利用一道近紅外光的雷射聚焦在空氣與液體介面上。透過這項新穎的方法，可以達到空間及時間上控制的結晶化，還可能控制結晶的晶形結構。甚至在未飽和溶液中，光壓(雷射捕捉)可提高局部的溶液濃度至過飽和，使結晶化成功。這項新穎的結晶化被嘗試應用在許多化合物上，但並不是每一個化合物都能成功，而且它的機制尚未明瞭。在這研究中，我們探討雷射捕陷結晶化動力學與其機制，基於研究與觀察脯氨酸雷射捕陷結晶化於不同的溶劑中。首先我們發現脯氨酸於重水中，雷射捕陷結晶化不易成功。藉由檢驗不一樣的溶劑至乙醇(EtOD)，我們最終成功地首次示範出脯氨酸的雷捕陷結晶化並且在結晶化前發現了其高濃度液滴的形成。高濃度液滴是一種液態/液態的相分離，藉由觀察它的形成使我們得以進一步探究在光壓下分子聚集的機制與原理。最後，根據觀察到雷射捕陷所引發的現象，我們將討論許多因素(溶劑的性質，溶質與溶劑分子的作用力與雷射引發局部溶液環境的改變)對於雷射捕陷結晶化和高密度液滴形成造成的影響，這些將助於我們對於雷射捕陷聚集化和結晶化有更深的認識。

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Acknowledgement

I am very lucky and appreciate that I had chance to join this great group. Actually I stay here less than 1.5 years but the experience is so beautiful that I can not forget forever. “Global village”, this word is really touched me deeply after I entering this lab. Until now, sometimes I still feel unbelievable that I work with so many foreign friends. The times here is really enjoyable and I thank everything here.

I have to appreciate to my supervisor, Prof. Miura. We have the most close and frequent contacting. Sincerely thanks to his teaching and guiding with patience and kindness, it inspired me so much that I can finish my study. I have no word to express my gratitude to him.

Thank to Prof. Masuhara. His scientific consideration and sense are stimulated and elevated to me. I am very glad and proud that I can study with him.

I also want to thank Prof. Sugiyama that he gives me lots useful suggestions. His great supporting let me clarify my research more deeply. Dr. Usman and Dr. Uwada teach me a lot and help me of experimental step up for Raman measurement. I am very grateful for their help.

Besides, I would like to thanks to all members of Masuhara group in NAIST, especially to Mr. Iino, Dr. Rungsimanon, Dr. Okano, Dr. Maezawa. They give me an impressive

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memory in Japan. Additionally thanks to Dr. Yuyama, we had many discussion and that improve my understanding of my study.

I must have to thanks my seniors and classmates: 許平諭, 杜靜如, 李依純, 劉宗翰, 黃彥樺, 曾綮續, 許孜瑋, 王順發, 江威逸, 黃鈴婷。Thanks for their companying with nice atmosphere and encouragement to me when I was disappointed. Specially thanks to 許平諭, 杜靜如 and 劉宗翰, they help me a lot not only for daily life but also research. Thanks to 李依純, she introduced me into this group. Without her, I could not join this great group. Wish them will get great achievement in the future.

Finally, sincerely thanks to my family for their mentally and financially concerning and support to finish master degree.

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List of Figures

1. Introduction ... 1

Fig. 1.1 Ray diagram shows difference of the direction of gradient force (gray arrows) induced by unfocused (a) and focused (b) light. ... 3

Fig. 1.2 Schematic drawing of the relationship between refractive indices of object (n1) and medium (n2) and the direction of gradient force. ... 3

Fig. 1.3 Schematic view of PNIPAM assembly via photon pressure and phase transition ... 5

Fig. 1.4 Schematic picture of possible convection flow and trapped molecules brought up by Uwada et al. ... 6

Fig. 1.5 Schematic drawing of nucleation, molecules start to pack with each other. ... 11

Fig. 1.6 Phase diagram showing the solubility depends on temperature and concentration. ... 11

Fig. 1.7 Free energy diagram for possible crystallization processes. Curved depict phase transition from liquid, dense liquid as intermediate state, and crystalline phases. Black broken line indicates the free energy for liquid-liquid phase separation. Red and blue broken lines indicate the energy of droplet with (a) lower and (b) higher free energy than that of initial liquid phase. ... 12

2. Experimental ... 20

Fig. 2.1 Solution height distribution in (a) cover glass and (b) bottom glass dish ... 22

Fig. 2.2 Schematic diagram of optical set up of laser trapping crystallization system. ... 23

Fig. 2.3 Schematic diagram of optical set up of laser scanning confocal microscope. ... 24

Fig. 2.4 Chemical structure of proline. (a) Neutral and (b) zwitterionic form ... 25

Fig. 2.5 (a) Crystal structure of L-proline and (b) hydrogen-bonded dimer structure formed between the columns in a crystal. ... 27

Fig. 2.6 Proposed model of the molecular arrangement of L-proline and water in a saturated proline solution. ... 27

Fig. 2.7 Optical path length dependence of transmittance of (a) EtOD and EtOH, (b) D2O and H2O and (c) L-proline aqueous solution. ... 30

3. Laser Trapping Crystallization of L-proline in Deuterated Water (D2O) ... 33

Fig. 3.1 (a) Power and (b) initial solution height dependent local solution height change. Supersaturated value was 0.9 SS. Power dependence measured with 0.5, 0.7, 0.9, 1.0 W and no laser irradiation. Lower panel shows initial solution height dependence measured with 30, 60, 80, 85 and 95 m. Laser power was 1.0 W. ... 34

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Fig. 3.2 Further irradiation, solution height was decreased to near the bottom glass substrate and then gave two results (a) Crystallization (b) Local totally dry, absence of

solution ... 35 Fig. 3.3 Crystals induced (a) by focused irradiation and (b) by solvent evaporation. ... 36 Fig. 3.4 (a) Concentration and (b) power dependence on crystallization probability of

L-proline in D2O. Concentrations were 0.83, 0.88, 0.93 0.98 and 1.03 SS under

fixed laser power (1 W). Power dependence measured at 0.8, 1.0 and 1.2 W. Supersaturated value was 0.83 SS. Raw values of crystallization probability were mentioned at the bottom each bar. ... 37 Fig. 3.5 Local solution surface height change of neat D2O solution by giving 1 W laser to

the air/solution interface ... 38 Fig. 3.6 Schematic representation of local dry spot formation. (a) Initial solution without

laser irradiation. (b) Focusing trapping laser to the air/solution interface causes sinking of the surface by surface tension decreasing and convection flow around the laser spot. (c) Proline and solvent molecules flow into focal spot with

convection flow. A few proline molecules are trapped, but most of them are flowed away. (d) Continuous change of the solution height makes surface reached to the substrate surface and finally solution is spread to the surroundings and forms a dry spot. ... 40 Fig. 3.7 Illustration of proposed crystallization process. (a) Initial solution before laser

irradiation. (b) Focus the trapping laser to the air/solution interface and trap small numbers of proline molecules. (c) Thermal capillary force and drastic decrease in space resulting in an accumulation of molecules. (d) Crystallization. (c) and (d) are magnified view around the focal point. ... 42 Fig. 3.8 (a) Initial crystal shape before laser irradiation and (b) after started irradiation.

Irradiation caused dissolution of crystal. (c) Crystal formation after terminating laser irradiation. (d) Further growth of new crystal. Slight curvature of growth front line is considered due to the dissolution. Yellow dashed lines indicate original

shape before changing irradiation condition. ... 44 Fig. 3.9 (a) Initial crystal shape (b) Laser on, induced crystal growth mainly along the

original direction. (c) and (d) show further crystal growth and its local dissolution near laser spot ... 45

4. Laser Trapping Crystallization of L-proline in Mixed Solvent (D2O & EtOD) ... 48

Fig. 4.1 Solution height change recorded during irradiation of 1 W trapping laser. ... 49 Fig. 4.2 Mixed solution (a) before and (b) after trapping laser irradiation. Solution size was

shrunk in (b). ... 50

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Fig. 4.3 Pictures of crystal formation and growth. (a) Before crystallization, (b) 2 s, (c) 6 s, and (d) 12 s after starting crystallization. Quite rapid crystal growth was observed. 50

5. Laser Trapping Crystallization of L-proline in Deuterated Ethanol (EtOD) ... 54 Fig. 5.1 Local solution height change during laser irradiation. Height was first lowered and

becoming thin. After kept very thin condition, height was elevated and eventually showed crystal formation. Above curve was obtained with 1.2 SS solution. Laser power: 1.0 W. ... 56 Fig. 5.2 Boundary of droplet observed under microscope. (a) Initial droplet formation at the

focal spot, (b) droplet located at the corner of screen and (c) moving of boundary due to an expansion of droplet. ... 56 Fig. 5.3 Bright field image of proline crystal induced by focused laser after solution height

elevation started. Zero second corresponds to 5 min in Fig 5.1. Crystal formed from 1.2 SS solution. Laser power was 1.0 W. ... 57 Fig. 5.4 Local solution height change by trapping laser irradiation. Solvent dried and it did

not show surface elevation. Crystal was formed without height elevation.

Supersaturated value was 1.0 SS. Applied laser power was 1.0 W. ... 58 Fig. 5.5 Typical crystal structure formed without solvent height elevation. ... 58 Fig. 5.6 (a) Probability of crystallization without solution surface elevation. Sample is

proline/EtOD. Supersaturated value is 1.0~1.5 SS. (b) Schematic drawing of laser power dependent number of trapped molecules before rupturing solution layer and crystallization. Red and blue lunes indicate necessary time to rupture solution layer and optical trapping rate under photon pressure, respectively. (c) power dependent number of trapped molecules after considering two factors depicted in (b). ... 59 Fig. 5.7 Concentration (a) and trapping power (b) dependence on crystallization probability

of proline in EtOD. Applied laser power for concentration dependence was 1.0 W. Supersaturated value of solution used for power dependence was 1.3~1.4 SS. ... 61 Fig. 5.8 The distribution of solution height at the moment of crystallization for (a) single

needle-like crystal and (b) polycrystal. 0.7 ~1.5 SS proline/EtOD solution and 0.6 ~1.2 W laser power used. ... 63 Fig. 5.9 Crystallization process in dense liquid droplet by laser trapping crystallization. (a)

Molecular clusters and aggregates in droplet. Interaction is not so strong. (b)

Condensation and collection of them by photon pressure. (c) Crystallization. ... 65 Fig. 5.10 (a) Bright field image of crystal formed by laser trapping. Image taken 5 sec after

crystallization started and (b) corresponding crossed Nicole image of (a). (c)

Crossed Nicole image of the same crystal grown by laser trapping for 1 min. ... 67

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Fig. 5.11 SHG signal observed during trapping crystallization. Light source for SHG and laser trapping was the same 1064 nm laser beam. Trapping laser power is 1.0 W. Green spot in the image is SHG. Blue spot is 488 nm light from diode laser. ... 68 Fig. 5.12 Raman spectra of proline crystal formed by laser trapping crystallization (red),

commercial powder (black), and crystalline proline data from reference [13](blue). Table inside figure is assignment of the peaks which is referred from reference [13].

... 69

6. Dense Liquid Droplet ... 73 Fig. 6.1 Illustration represents observed droplet formation process. (a) Irradiation to the

air/solution interface. (b) Surface deformation due to Marangoni effect and

convection flow induced. (c) Efficiently collecting molecule through mass transfer and higher concentration phase was formed. (d) Grown droplet; large

concentration different between droplet and outside of it resulting in liquid-liquid phase separation. ... 74 Fig. 6.2 Picture shows millimeter-size liquid droplet. White arrow indicates the place of

droplet. ... 74 Fig. 6.3 Dissolution of crystals near phase boundary of droplet after shut-down of trapping

laser. Crystals before (a) and after dissolving (b). Black arrows indicate phase

boundary. ... 76 Fig. 6.4 Comparison of local height change during laser trapping between glycine in D2O

and proline in EtOD. Same sample container, cut sample vial, was used for both sample. ... 77 Fig. 6.5 Backward scattering intensity from the air/solution interface reconstructed from

EMCCD image during laser trapping. ... 79 Fig. 6.6 Schematically illustrating the droplet formation and crystallization based on

concentration and molecule alignment. ... 81 Fig. 6.7 Phase diagram showing temperature and concentration dependent phase transition.

Arrow indicates transition path from liquid to liquid-liquid separation phase. ... 82 Fig. 6.8 Schematic drawing of free energy for droplet formation in different solvent ... 84

7. Summary ... 87

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List of Tables

Table 2-1 Property of D2O and EtOD, and solubility of L-proline of them ... 26

Table 2-2 Absorption coefficient (1064 nm), thermal conductivity and estimation of

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1. Introduction

1.1 Laser trapping

Laser trapping has been a well-known and powerful tool for its wide application specially in biology, chemistry and physics. It can manipulate from micrometer-sized to a few tens nanometer-sized objects freely without any mechanical contact by highly focusing incident light. We have applied this technique to assemble molecules in this work.

1.1.1 History

The pioneer of laser trapping experiment, Arthur Ashkin et al., found the possibility of optical trapping of transparent micron-sized particle, and finally they first demonstrated it in 1970 [1]. They reported that they could detect gradient force and scatter force when the laser beam is tightly focused on the particle.

It was not known that focused light can generate stable three-dimensional optical trap at that time. Later, Ashkin confirmed the single-beam gradient force by utilizing optical trapping method [2] and this technique has been successfully applied to the wide range of particles from dielectric to biological ones.

Chu and his colleague extended Ashkin’s method to trap atoms, and he received the 1997 Nobel prize in physics along with Claude Cohen-Tannoudji and William Daniel Phillips by

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the work of laser cooling and trapping of atoms [3]. Indeed it was a very big breakthrough to control atom in the 1 Å scale. Furthermore, living cells such as bacteria can be manipulated without damage [4].

Until nowadays, this technique has been continuously applied to the wide fields of physical and biological studies. Usually laser trapping is applied to single micrometer-sized particles and typically to nanoparticles, and now being developed to combine with single molecule spectroscopy [5, 6].

1.1.2 Principle of laser trapping

Laser trapping, the physical phenomenon, is due to the interaction between light and target objects. Traditionally, optical force is classified into two parts: gradient force and scattering force. The former is directed along the spatial laser gradient and the latter is along the direction of light propagation. For stable trapping of objects in three dimensional space, the gradient force which transfers the object to the focal region must be larger to exceed the scattering force which moves the object away from the focal region. This condition is provided when very sharp light intensity change is achieved by using an objective lens with high N.A.

By assuming that the object is sphere and the size is much larger than the wavelength of trapping light, Mie scattering theory holds and the gradient force can be interpreted by Ray optics (Fig. 1.1) [7]. Since the refraction of incident light takes place when light passed

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through the object, momentum is transferred from photon to object. According to the Newton’s third law, the changed momentum of photon and that of object should be equal. Then, if the refractive index of the trapped object is higher than that of the medium, finally the object is moved to the focused spot as depicted in Fig. 1.2a [8]. Contrary, if the refractive index of trapped object is lower than that of medium, the direction of optical force is opposite as seen in Fig. 1b.

Fig. 1.2 Schematic drawing of the relationship between refractive indices of object (n1) and

medium (n2) and the direction of gradient force.

Fig. 1.1 Ray diagram shows difference of the direction of gradient force (gray arrows) induced by unfocused (a) and focused (b) light.

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On the other hand, Raleigh scattering is dominant in the case of trapping of small spherical object which is much smaller than the wavelength of trapping light. Under this condition the object is considered as a dielectric particle, i.e. point dipole. We need to consider the interaction between an electric field of the light and dipole moment of the particle. Gradient and scattering forces are corresponding to the first and second term of equation 1.1, respectively, where E is electric field, and B is magnetic field. An equation 1.2 depicts α which is the polarizability of a particle to be trapped, where r is the radius of the particle, and

ε2 is the dielectric constant of the surrounding medium. n1 and n2 are the refractive indices of

the particle and the surrounding medium, respectively.

………... (1.1)

……… (1.2)

As the high N.A. objective lens is employed, the trapping potential becomes to the equation (1.3). Once the trapping potential overcomes the Brownian motion whose energy should be

kBT, where kB is the Boltzmann constant and T is the temperature in Kelvin, photon pressure

makes it possible to control the object.

……….…….……… (1.3)

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the refractive index of the object should be larger than that of medium (n1 > n2)

1.1.3 Laser trapping-induced assembly formation

Different from single particle manipulation by laser trapping, application of this technique allowed scientists to investigate the interaction of numbers of particles such as colloids [9], polymers, and membranes [10]. Additionally, it can be applied to collect molecule. Indeed it has been demonstrated to assemble small particles of colloids and polymers [11, 12] to create their assembly which is as large as the focal spot size. Fig. 1.3 depict schematic representation of assembly formation of PNIPAM (Poly(N-isoprpryl acrylamide)) that induced by focusing of trapping light source into the solution [12]. Masuhara et al. have investigated the assembly formation of plenty polymer molecules under photon pressure [13-15] and its solvent dependence [16].

Fig. 1.3 Schematic view of PNIPAM assembly via photon pressure and phase transition [12].

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On the other hand, heat generation by absorption of focused light is inevitable in laser trapping. It induces Marangoni convection [17, 18] and enhances mass transfer [19] that increases molecular transportation which should cooperate with photon pressure to collect molecules. As we can see in the phase transition of PNIPAN [12], we describe this matter in the last paragraph. Recently the convection flow under trapping could be realized in Fig. 1.4 by T. Uwada et al. [20].

Above mentioned observation of molecular assembly formations induced by laser trapping imply a possibility of more advanced molecular assembling, i.e. well-ordered molecular assembly such as crystallization should be possible. Tsuboi et al. confirmed the assembling of several amino acids by observing their Raman scattering spectra and backward scattering [21]. They explained that laser trapping induced assembly is probably due to trapping clusters of solute molecules. Lysozyme was the first successful example of

Fig. 1.4 Schematic picture of possible convection flow and trapped molecules brought up by Uwada et al. [20].

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laser trapping, but crystallization was observed at few days after the focused laser irradiation [22]. It reported crystallization could be induced by photon pressure causing protein aggregate. Besides, in the same proteins, lysozyme, W. Singer applied trapping to induce the crystal growth [23] and investigated directional of crystal growth under trapping [24, 25].

1.1.4 Laser trapping crystallization

Sugiyama et al. demonstrated glycine crystallization induced by laser trapping in 2007 [26]. It was the first observation of the crystallization only by focused irradiation. They named this method as “laser trapping crystallization”. It is not only a new application of laser trapping but also novel methodology of crystallization.

In general, where and when crystallization took place is not clear, but now it is always observed at the focal spot via laser trapping within a few minutes. Moreover, they reported spatially-controlled crystal growth [27] and molecular orientation in crystal, as direction of crystal growth was directed toward laser spot and different polymorph of glycine crystal were prepared by adjusting laser power [28].

Initially, laser trapping crystallization was reported for supersaturated glycine solution. Usually it is impossible to crystallize molecules in unsaturated solution, but they can observe crystallization even in unsaturated solution by laser trapping crystallization [29]. It

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suggests that local concentration increase to supersaturated value in the laser spot due to laser trapping of the clusters, leading to crystallization.

Besides, laser trapping crystallization showed not only spatiotemporally controlled crystallization but also very interesting photon pressure-induced phenomenon; large liquid droplet formation. Yuyama et al. demonstrated millimeter-sized dense liquid droplet formation of glycine. They observed the droplet formation by focusing the trapping laser to the solution/substrate interface [30]. Moreover, glycine crystallization was observed immediately after when the focus of trapping laser moved to the air/solution interface. It implies higher-concentration droplet formation, i.e. local concentration elevation due to the photon pressure, and they concluded that the dense droplet would be a precursor of the crystal.

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1.2 Crystallization

We study crystallization induced by laser trapping. Crystallization is closely related to our daily life such as the salt isolation from seawater and diamond formation in the deep earth under high temperature and pressure. Crystal provides molecule packing information and is used in wide fields of science and technology.

1.2.1 History and study of biomolecular crystallization

According to the history of crystallization described by McPherson [31], it can be traced back to more than 150 years ago. The first published observation of crystallization was reported by Hunefeld on hemoglobin in 1840 [32]. They reported the crystallization of blood of earthworm when it was pressed between two microscope slides. And later studies, Sumer and Stanley were awarded the Nobel Prize for chemistry in 1946 by isolation and crystallization of proteins and viruses [33]. In addition, it is also significant to analyze the structure of crystal to clarify functions of biomolecules. The first structural determination of biomolecule has done for vitamin B-12 in 1957 by D. C. Hodgkin by using its crystal [34]. She received the Novel prize for chemistry as the result of this research.

Crystallization has been studied for long time, however, its process is very complex and crystallization of some large biomolecules is still very difficult. In order to understand the fundamentals of crystallization to obtain better crystals, massive efforts were made on

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optimized crystallization and crystal growth of basic molecules such as small organic compounds and amino acids. Crystallization of amino acids, giving basic information to understand it, is still important works for protein crystallization.

This is one of the reasons why amino acids were employed in this work. Although crystallization is still quite empirical, further understanding of crystallization can be expected.

1.2.2 Crystallization theory

Crystallization is a phase-transition phenomenon and also widely used as a purification method. Usually, solution for crystallization must be under supersaturation, and for achieving supersaturated solution there are many variable methods such as vapor pressure, temperature and pH valve. Crystallization process can generally be separated into two parts of nucleation and crystal growth processes. The birth of a new crystal is called nucleation: it indicates that molecule aggregate becomes larger than the critical size. Traditionally, the classical nucleation theory has been employed for the nucleation process, but it starts with tiny size and it is difficult to observe experimentally (Fig. 1.5) [35]. Many papers have studies the nucleation and its mechanism in detail [36, 37]. After nucleation, a subsequent process is known as crystal growth where nuclei grow larger. Molecules are continuously packing with each other in the regular ordering.

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The process of crystallization also can be schematically illustrated by phase diagram (Fig. 1.6). The diagram is well interpreted with influential parameters of crystallization such as concentration and temperature. Under this condition, solution would be divided into three parts depending on the solution saturation. Once solution became highly saturated with higher free energy in the labile or metastable region, nucleation could take place, causing a reduction of free energy and the phase returned to the stable region. As previously mentioned, we can suppose the dense liquid is regarded as precursor of crystallization in the intermediate state as seen in Fig. 1.7 [34].

Crystallization Temperature S ol uti on concentr ati on

Fig. 1.6 Phase diagram showing the solubility depends on temperature and concentration.

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1.2.3 Laser-induced crystallization

Laser has been developed since 1960. It provides very stable monochromatic light and has been applied to wide fields of chemistry, physics, biology, materials science. There are plenty of researches using lasers. Crystallization is not the exception.

There are conventional methods to crystallize such as Batch crystallization and vapor diffusion. Laser-induced crystallization has been received attention and developed because it could regularly generate crystals or control the initial orientation giving different morphology of crystals, even finding novel crystal structure. Moreover, in this work, spatiotemporally controlled control crystallization could be achieved by laser trapping. Following is the introduction of laser induced crystallization.

Fr

ee ene

rgy

G

Nucleation reaction coordinate

Solut ion De nse liqu id Cr ystals Solut ion De nse liqu id Cr ystals (a) (b)

Fig. 1.7 Free energy diagram for possible crystallization processes. Curved depict phase transition from liquid, dense liquid as intermediate state, and crystalline phases. Black broken line indicates the free energy for liquid-liquid phase separation. Red and blue broken lines indicate the energy of droplet with (a) lower and (b) higher free energy than that of initial liquid phase.

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1.2.3.1 Photochemical reaction induced crystallization

In general, laser induced crystallization can be divided into two parts, photochemically and optically induced crystallization. The early work of the former part, John Tyndall has studied in a range of vapors and solutions in 1869 [38]. Instead of laser just irradiation with conventional lamps also could achieve photochemical crystallization [39].In this method of crystallization, the light with high energy is enough to cause ionization or create radicals and subsequent reactions induce nucleation.

1.2.3.2 Optical crystallization

For the latter one, optically induced nucleation was discovered first by Garetz et al. in 1996 [40]. In their work, supersaturated solution of urea was irradiated by 20 ns pulse of 1064 nm laser light with energy of about 0.1 J per pulse. It is considered as nonphotochemical reaction because the power and wavelength of light were not able to cause photochemical reaction.

The result showed polarization dependent orientation of crystallite of urea where molecules were oriented along the incident light polarization. Authors suggested that this phenomenon was probably caused by the optical Kerr effect. Besides, following their discovery, most notably case, α- and γ- polymorphs of glycine crystals were induced from solution by circular and linear polarization of light, respectively; it is mentioned as

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polarization switching [41]. Polarization switching could be made possible by the matching between packing arrangements of molecule and polarization of light. For example, α-glycine is composed of cyclic dimmers. Meanwhile γ-glycine is composed of helical chains. Recently, polarization switching was also reported in case of L-histidine [42].

Instead of Kerr effect mechanism, other nonphotochemical laser-induced crystallization method had been also developed. For example, femtosecond laser induced crystallization through bubble formation [43], single pulse crystallization via mechanism for the effect involves the isotropic electronic polarization of cluster [44] and specially this work, laser trapping crystallization.

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1.3 Motivation

We are interested in molecule assembly formation and crystallization induced by photon pressure. Since for only laser trapping crystallization just succeeded in limited number of molecules and its behavior is not totally clarified, more extension of experiments involving other amino acids have been tried until now. Indeed the spatiotemporal control of crystallization through laser trapping crystallization is quite important technique. We need to understand dynamics and mechanism of molecular crystallization under photon pressure to establish this method as a general crystallization technique.

In this work, we intend to investigate the influence of different solvents so that solutions of L-proline in D2O and EtOD were applied as sample. This would be the indication in the

solvent selection. The interaction of solute with solvent affects molecule assembling under photon pressure, which was discussed base on our observation. It may imply what kind environment is efficient to allow molecules trapped, then to crystallize them, and more favorably to get their nice crystals.

Not only developing this technology but also investigating crystallization process could be extended more. As the precursor of crystallization, dense liquid induced by irradiation can also be demonstrated. We hope more understanding and extension of laser tapping crystallization can be achieved through this work.

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1.4 References

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Review Letters, 1970. 24: p. 156-159.

2. A. Ashkin., J.M. Dziedzic, J.E. Bjorkholm, and S. Chu, Observation of a

single-beam gradient force optical trap for dielectric particles. Optics Letters, 1986.

11: p. 314-317.

3. S. Chu, J.E. Bjorkholm, A. Ashkin, and A. Cable, Experimental Observation of

Optically Trapped Atoms. Physical Review Letters, 1986. 57: p. 314-317.

4. A. Ashkin, J.M. Dziedzic, and T. Yamane, Optical trapping and manipulation of

single cells using infrared laser beams. Nature, 1987. 330: p. 314-317.

5. M.J. Lang, P.M. Fordyce, and S.M. Block, Combined optical trapping and

single-molecule fluorescence. Journal of Biology, 2003. 2: p. 6.1-6.4.

6. M.A.v. Dijk, L.C. Kapitein, J.v. Mameren, C.F. Schmidt, and E.J.G. Peterman,

Combining Optical Trapping and Single-Molecule Fluorescence Spectroscopy Enhanced Photobleaching of Fluorophores. J. Phys. Chem. B, 2004. 108: p.

6479-6484.

7. K.C. Neuman and S.M. Block, Optical trapping. Review of Scientific Instruments, 2004. 75: p. 2787-2809.

8. K. Sasaki, M. Koshioka, H. Misawa, N. Kitamura, and H. Masuhara, Optical

trapping of a metal article and a water droplet by a scanning laser beam. Appl.

Phys. Lett., 1991. 60: p. 807-809.

9. J.C. Crocker and D.G. Grier, When Like Charges Attract: The Effects of Geometrical

Confinement on Long-Range Colloidal Interactions. Physical Review Letters, 1996.

77: p. 1897-1900.

10. R. Bar-Ziv and E. Moses, Instability and "Pearling" States Produced in Tubular

Membranes by Competition of Curvature and Tension. Physical Review Letters,

1994. 73: p. 1392-1395.

11. S.C, Chapin, V. Germain, and E.R. Dufresne, Automated trapping, assembly, and

sorting with holographic optical tweezers. Optics Express, 2006. 14: p.

13095-13110.

12. J. Hofkens, J. Hotta, K. Sasaki, H. Masuhara, and K. Iwai§, Molecular Assembling

by the Radiation Pressure of a Focused Laser Beam: Poly(N-isopropylacrylamide) in Aqueous Solution. Langmuir, 1997. 13: p. 414-419.

13. P. Borowicz, J.-i. Hotta, K. Sasaki, and H. Masuhara, Chemical and Optical

Mechanism of Microparticle Formation of Poly(N-vinylcarbazole) in N,N-Dimethylformamide by Photon Pressure of a Focused Near-Infrared Laser

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Beam. J. Phys. Chem. B, 1998. 102: p. 1896-1901.

14. J.-i. Hotta, K. Sasaki, and H. Masuhara, Laser-Controlled Assembling of Repulsive

Unimolecular Micelles in Aqueous Solution. J. Phys. Chem. B, 1998. 102: p.

7687-7690.

15. T.A. Smith, J.-i. Hotta, K. Sasaki, H. Masuhara, and Y. Itoh|, Photon

Pressure-Induced Association of Nanometer-Sized Polymer Chains in Solution. J.

Phys. Chem. B, 1999. 103: p. 1160-1163.

16. P. Borowicz, J.-i. Hotta, K. Sasaki, and H. Masuhara, Laser-Controlled Association

of Poly(N-vinylcarbazole) in Organic Solvents: Radiation Pressure Effect of a Focused Near-Infrared Laser Beam. J. Phys. Chem. B, 1997. 101: p. 5900-5904.

17. M. Gugliotti, M.S. Baptista, and M.J. Politi, Laser-Induced Marangoni Convection

in the Presence of Surfactant Monolayers. Langmuir, 2002. 18: p. 9792-9798.

18. Z.-S. MaoI and J. Chen, Numerical simulation of the Marangoni effect on mass

transfer to single slowly moving drops in the liquid-liquid system. Chemical

Engineering Science, 2004. 59: p. 1815-1828.

19. O.A. Louchev, S. Juodkazis, N. Murazawa, S. Wada, and H. Misawa, Coupled laser

molecular trapping,cluster assembly, and deposition fed by laser-induced Marangoni convection. Optics Express, 2008. 16: p. 5673-5680.

20. T. Uwada, T. Sugiyama, A. Miura, and H. Masuhara, Wide-field light scattering

imaging of laser trapping dynamics of single gold nanoparticles in solution. Proc. of

SPIE, 2010. 7762: p. 77620N-1~8.

21. Y. Tsuboi, T. Shoji, and N. Kitamura, Optical Trapping of Amino Acids in Aqueous

Solutions. J. Phys. Chem. C, 2010. 114: p. 5589-5593.

22. Y. Tsuboi, T. Shoji, and N. Kitamura, Crystallization of Lysozyme Based on

Molecular Assembling by Photon Pressure. Japanese Journal of Applied Physics,

2007. 114: p. L1234-L1236.

23. W. Singer, U.J. Gibson, T.A. Nieminen, N.R. Heckenberg, and H. Rubinsztein-Dunlop, Towards Crystallization using Optical Tweezers. Proc. of SPIE, 2006. 6038: p. 60380B-1~8.

24. W. Singer, T.A. Nieminen, U.J. Gibson, N.R. Heckenberg, and H. Rubinsztein-Dunlop, Orientation of optically trapped nonspherical birefringent

particles. Physical Review 2006. 73: p. 1-5.

25. W. Singer, H. Rubinsztein-Dunlop, and U. Gibson, Manipulation and growth of

birefringent protein crystals in optical tweezers. Optics Express, 2004. 12: p.

6440-6445.

26. T. Sugiyama, T. Adachi, and H. Masuhara , Crystallization of Glycine by Photon

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27. T. Sugiyama, T. Adachi, and H. Masuhara, Crystal Growth of Glycine Controlled by

a Focused CW Near-infrared Laser Beam. Chemistry Letters, 2009. 38: p. 482-483.

28. T.Rungsimanon, K.-i. Yuyama, T. Sugiyama, H. Masuhara, N. Tohnai, and M. Miyata, Control of Crystal Polymorph of Glycine by Photon Pressure of a Focused

Continuous Wave Near-Infrared Laser Beam. J. Phys. Chem. Lett., 2010. 1: p.

599-603.

29. T. Rungsimanon, K.-i. Yuyama, T. Sugiyama, and H. Masuhara, Crystallization in

Unsaturated Glycine/D2O Solution Achieved by Irradiating a Focused Continuous

Wave Near Infrared Laser. Crystal Growth & Design, 2010. 10: p. 4686-4688.

30. K.-i. Yuyama, T. Sugiyama, and H. Masuhara, Millimeter-Scale Dense Liquid

Droplet Formation and Crystallization in Glycine Solution Induced by Photon Pressure. J. Phys. Chem. Lett., 2010. 38: p. 1321-1325.

31. A. McPherson, Crystallization of biological macromolecules. 1999, New York, USA

Cold Spring Harbor Laboratory Press.

32. F.L. Hünefeld, Der Chemismus in der thierischen Organisation, 1840: p. 158-163. 33. W.M. Stanley, Isolation of a crystalline protein possessing the properties of

tobacco-mosaic virus. Science, 1935. 81: p. 644-645.

34. D.C. Hodgkin, J. Kamper, J. Lindsay, M. MacKay, J. Pickworth, J.H. Robertson, C.B. Shoemaker, J.G. White, R.J. Prosen, and K.N. Trueblood, The Structure of

Vitamin B12 I. An Outline of the Crystallographic Investigation of Vitamin B12. Proc.

R. Soc. Lond. A, 1957. 242: p. 228-263.

35. P.G. Vekilov, Dense Liquid Precursor for the Nucleation of Ordered Solid Phases from Solution. Crystal Growth & Design, 2004. 4: p. 671-685.

36. J.M. Garcı´a-Ruiz, Nucleation of protein crystals. Journal of Structural Biology, 2003. 142: p. 22-31.

37. A.A. Chernov, Protein crystals and their growth. Journal of Structural Biology, 2003.

142: p. 3-21.

38. J. Tyndall, Philos. Mag. , 1869. 37: p. 384.

39. S.p. Veesler, K. Furuta, H. Horiuchi, H. Hiratsuka, N. Ferte, and T. Okutsu, Crystals

from Light Photochemically Induced Nucleation of Hen Egg-White Lysozyme.

Crystal Growth & Design, 2006. 6: p. 1631-1635.

40. B.A. Garetz, N. J. E. Aber, L. Goddard, R.G. Young, and A.S. Myerson,

Nonphotochemical, Polarization-Dependent, Laser-Induced Nucleation in Supersaturated Aqueous Urea Solutions. Physical Review Letters, 1996. 77: p.

3475-3476.

41. B.A. Garetz and J. Matic, Polarization Switching of Crystal Structure in the

Nonphotochemical Light-Induced Nucleation of Supersaturated Aqueous Glycine Solutions. Physical Review Letters, 2002. 89: p. 177501-1~4.

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42. X. Sun, B.A. Garetz, and A.S. Myerson, Polarization Switching of Crystal Structure

in the Nonphotochemical Laser-Induced Nucleation of Supersaturated Aqueous L-Histidine. Crystal Growth & Design, 2008. 8: p. 1720-1722.

43. K. Nakamura, Y. Sora, H.Y. Yoshikawa, Y. Hosokawa, R. Murai, H. Adachi, Y. Mori, T. Sasaki, and H. Masuhara, Femtosecond laser-induced crystallization of protein in

gel medium. Applied Surface Science, 2007. 253: p. 6425-6429.

44. A.J. Alexander and P.J. Camp, Single Pulse, Single Crystal Laser-Induced

Nucleation of Potassium Chloride. Crystal Growth & Design, 2009. 9: p. 958-963.

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2. Experimental

2.1 Materials

D2O (>99%), EtOD (>99%), and L-proline (>99%) were obtained from Sigma-Aldrich

and used without any further purification. Concentration of D2O and EtOD solution of

L-proline is ranging 1.500 ~ 1.950 g/mL and 0.006~ 0.020 g/mL, respectively. Solute molecules in the solution were ensured to be totally dissolved by heating with a water bath to 60˚C for 8-12 h in the glass vial (Nichiden-Rika glass) and then the solution was left until it returned to room temperature (~25˚C). Prior to the laser trapping crystallization experiments solutions were aged for 1 to 7 days to ensure the absence of spontaneous crystallization. Here we used deuterated water and ethanol (D2O and EtOD, respectively) as

solvents to suppress temperature elevation due to trapping laser absorption. Details are discussed in section 2.5.

Since an experimental requirement to focus trapping laser light to the solution surface with a short-working distance objective lens, two different types of sample containers were mainly used in this study. First is a flat glass substrate. A cover glass (24 mm × 30 mm, Gold Seal) was mainly employed for EtOD solution of L-proline (Fig. 2-1a). In contrast, D2O solution of L-proline is highly viscous and difficult to be spread forming thin layer of

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achieve thinner thickness of proline/D2O solution, a bottom glass dish (Ibidi, μ-dish, 35mm

high and 2 cm diameter) was mainly used (Fig. 2-1b) as the second sample container. For additional experiments in section 6.2, we use a home-made closed glass container which was fabricated by cutting glass vial (Nichiden-Rika glass) and glued on a cover slip by silicon glue (Shin-Etsu Silicone, 1 component RTV).

A shape of solution surface depends on what sample container is used. The surface shape of flat cover glass slip was convex and other two (bottom glass dish and cut glass vial container) were concave. Shapes and a distribution of solution thickness on different containers were depicted Fig. 2.1. Solution height distribution of cut glass vial container is similar to that of bottom glass dish. Applied volume of the solution was changed to adjust an initial solution thickness at the center to be about 70~150 m for all conditions.

Before usage of the containers, all containers were washed with detergent, acetone and purified water repeatedly. Washed containers were further cleaned by dry washing method with applying oxygen plasma treatment (10 minutes with oxygen gas flow rate of 40 cc/min). After wet and dry cleanings surface of all glass containers became highly hydrophilic and clean. Applied solution to clean and hydrophilic surface spreads and covers whole glass surface stably.

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2.2 Microscope set up: Imaging and spectroscopy

Fig. 2.2 schematically shows a microscope setup used in time-resolved laser trapping crystallization imaging and dynamics study. Microscope setup is based on an inverted microscope (Olympus, IX71). Room temperature and humidity were controlled to be around 23~25˚C and 50~60%, respectively. Linearly polarized near-infrared continuous wave 1064 nm Nd:YVO4 laser (Coherent, Matrix CW) was employed as a trapping light

source. The trapping laser was introduced into the microscope and focused to the air/solution interface through a 40× objective lens (N.A. 0.95). In order to achieve optimal trapping condition, trapping laser light was expanded and collimated to fully use pupil diameter of a microscope objective lens (~8 mm).

The other laser, 488 nm CW diode laser (Spectra-Physics, model name, 20 mW) was also

Solution height (μm) Distance (mm) (a) Cover glass 24 mm 30 mm Solution height (μm) Distance (mm) (b) Bottom glass dish 1 cm

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introduced into the microscope coaxially with a trapping NIR laser to check solution surface and a focusing position of NIR laser. It was also used as a scattering light source in the backward scattering measurement by collecting its reflection light with EMCCD camera.

Crystallization behavior near the laser spot was monitored and recorded by CCD or EMCCD camera. Bright field transmission imaging has carried out by using a halogen lamp as an illumination light source. Sample solutions were covered to suppress quick evaporation of solution during the measurement.

Confocal Raman scattering measurement was performed to characterize obtained crystals with an inverted microscope-based laser scanning confocal microscope (Olympus, FV300), where schematic diagram of confocal Raman system is shown in Fig. 2.3. A 532 nm DPSS laser (40 mW, JLW-532-200, SLOC) was employed as probe laser with the power ranger of

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30-40 mW.

Raman scattering signals were detected by cooled CCD camera (PIXIS400, Princeton instruments) combined with polychromator with a 150 grooves/mm grating (SpectraPro 2300i, Princeton instruments) through filter (Single-notch filter). We employed same objective lens (40×. N.A. 0.95) lens for Raman scattering measurement under laser trapping crystallization. All spectra were measured by integrating the signal for ~10 min. Proline crystals obtained by laser trapping crystallization in EtOD were buried in a transparent superglue to prevent a deliquescence by exposing to the water in the air.

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2.3 Characteristics of proline

Proline is one of the twenty proteinogenic amino acids. It is unique in amino acids. It has pyrrolidine ring as main framework and -amino group in its ring is secondary. Chemical structure of proline is depicted in Fig. 2.4 Proline can alternate its form from uncharged to the zwitterionic [1, 2]. Energetic difference of these two forms is negligibly small and structural transformation can occur smoothly.

The heterocyclic ring gives exceptional rigidity compared to the other amino acids despite its flexibility. Characteristic ring structure gives directionality in biological systems despite its conformational flexibility. Thus, proline is often found at the end of helix or in turns or loops in protein structure. Conformational flexibility of proline draws much attention since it is important for chemical reactivity and biological functionality for many molecules [1].

Proline is well-known as a highly hygroscopic compound. It suggests that proline strongly interacts with water molecule. It will break the intermolecular hydrogen bond between prolines easily [3] that confirmed from the Raman scattering spectra. Suppression

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of vibronic motions of carboxyl group on the proline molecule is interpreted due to the coordination of water molecule. It implies strong interactions between proline and water molecule [3-5]. According to its high affinity with water molecule, proline shows very high solubility in water (~1.6 g/mL, 23°C). Actually, addition of proline to some proteins can increase the solubility [5].Solubility of proline in different solvent [6, 7] applied in this study are is listed in table 2.1 with refractive indices and boiling points.

Table 2-1 Property of D2O and EtOD, and solubility of L-proline of them

D2O EtOD

Solubility of proline 1.9 g/ml (23°C) 0.012 g/ml (19 °C, EtOH)

Refractive index 1.33 (H2O) 1.36 (EtOH)

Boil point 100°C 79°C

The crystal structure of L-proline was first reported by Barbara et al. in 1949 [8]. Based on the hygroscopic character, it is difficult to form and grow water-free crystals and deterioration of crystals due to brief exposure to the atmospheric water vapor is not avoidable. The unit cell of the crystal was found to be orthorhombic with space group of P212121 and L-proline molecules were connected by intermolecular hydrogen-bonding as

shown in Fig. 2.5 [9]. Fig. 2.6 shows proposed molecular arrangement with taking into account of hydrogen bonding between water and proline based on crystal structure [5].

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2.4 Estimation of local temperature elevation induced by trapping laser

irradiation

Under laser trapping condition, temperature elevation due to laser absorption cannot be avoided. We should consider it since temperature change affects physical property and chemical reactivity of molecules in the solution. In general, temperature elevation causes increasing solubility of solute molecule and decreasing supersaturated value (SS). However,

Fig. 2.5 (a) Crystal structure of L-proline and (b) hydrogen-bonded dimer structure formed between the columns in a crystal.

Fig. 2.6 Proposed model of the molecular arrangement of L-proline and water in a saturated proline solution.

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temperature elevation enhanced evaporation of solvent and increasing of supersaturated value can occur simultaneously. Thus we need to estimate temperature elevation due to the absorption of trapping laser light at 1064 nm quantitatively.

Investigations of temperature increasing under laser trapping condition have been reported. In the case of 1064 nm CW YAG laser, Fischerreported that T/P ~5 K/W by thermal handing of langmuir-monolayers at the air/water interface [10]. Here T and P represent temperature in Kelvin and irradiated laser power in Watt. Schmid and Tromberg reported ~8 K/W and 10-14.5 K/W of temperature elevation of water, respectively [11-12]. Ito reported solvent dependent temperature elevation degree difference for ethylene, ethanol, and water as ~62±6, 49±7, and 23±1 K/W in ethylene, ethanol, and water, respectively, in small domain of solution base on the diffusion coefficient of fluorescent molecular determined by FCS [13].

Here heat is generated by irradiated laser light that was absorbed by solvent and solute molecules. First, we checked the absorption by solvent and solute molecules on the basis of Beer-Lambert’s Law,

where I0 and I are the intensity of incident and transmitted light, respectively. α and l are

absorption coefficient and optical path length. Absorption also can be defined based on molar absorption coefficient ε, optical length b, and concentration c.

  0e

l _ _

010

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Fig. 2.7 shows the optical path length dependent transmittance change of H2O, D2O,

EtOH, EtOD, and L-proline aqueous solution measured at 1064 nm. All the absorption and transmittance spectra were measured by absorption spectrophotometer (JASCO, V-600). As depicted in Fig. 2.7 optical path length dependent exponential decrease of transmittance was observed for all samples. Deuterated solvents showed smaller diminution of transmittance than that of H2O and EtOH. It indicates that deuterated solvents absorb less 1064 nm light

and, as a result, temperature elevation is smaller than others. Therefore we decided to use deuterated solvent to prevent temperature elevation in this research. By fitting the exponential curve based on the least square method, absorption coefficients of each solvent are as follows; H2O is ~ 14.5 m-1, D2O is ~1 m-1, and EtOH is ~11 m-1, and EtOD is ~4.6

m-1.

A contribution of L-proline molecule in an absorption of L-proline solution can be obtained from eq. 2-2, where x and y in the equation indicate solvent and proline respectively.

Molar absorption coefficient of proline at 1064 nm is obtained as ~ 5.0x10-3 M-1cm-1 from a mixture of 1.93M L-proline and 46.29 M water solution (Eq. 2-3). Compared to water, it is about five times larger than that of water (1.1x10-3 M-1cm-1).

      l

_

₁₀

xbxcx ybycy

e

………. (2-2)

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If we assume effective optical path length is represented by focal volume size dimension and we can estimate absorption by eq. 2-4 and the horizontal and lateral size of focal volume can be derived from egs. 2-5 and 2-6,

EtOD EtOH T ra ns m itted lig ht in te n sit y (a .u .)

Optical pass length (mm) (a) D2O H2O T rans mitte d light in tens ity (a.u. )

Optical pass length (mm) (b) L-proline aqueous solution (1.93 M) T ransmitte d light intens ity (a.u.)

Optical pass length (mm) (c)

Fig. 2.7 Optical path length dependence of transmittance of (a) EtOD and EtOH, (b) D2O

and H2O and (c) L-proline aqueous solution.

                 14 28 001

_

₁₀

113 103 10 46 29 pro 10 193

e

…….…... (2-3) 2 0

NA

n

4

1 Z











……….…….…………. (2-5) 3 2 z 2.3 d A 0 abs  _   _{……….……….. (2-4)}

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where r ,₀ Z ,₀ , NA, and n are the short and long axes, wavelength of light, numerical

aperture of the objective lens and refractive index of medium, respectively. Values of λ, NA, and n of solvents are known. Therefore r and ₀ Z are estimated to be 0.68 and 2.25 m, ₀ respectively.

Trapping laser light is tightly focused by high NA objective lens. If we consider only a central part contribute to the absorption and heat generation, the temperature elevation in the focal spot is represented by equation 2-7, where T, P and are temperature, laser power (W) and thermal conductivity, respectively [14].

Parameters in table 2-2 are substituted to eq. 2-7, and we can estimate the temperature elevation degree during laser trapping.

Table 2-2 Absorption coefficient (1064 nm), thermal conductivity and estimation of temperature elevation by irradiation (1064 nm) in different solvent

Sample ,absorption coefficient (m‐1) ,thermal Conductivity [W m‐1K‐1] Temperature elevation (△T/P) H2O 14.5 0.59 ~9.9 D2O 0.98 0.59 (H2O) ~0.7 EtOH 11.0 0.17 ~26.0 EtOD 4.6 0.17 (EtOH) ~10.9        0 5 w 0 61



r0 0 ….……..………. (2-6) 0 abs r 2 P T





   ………...………..……… (2‐7)

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2.5 References

1. J. Kapitan, et al., Proline Zwitterion Dynamics in Solution, Glass, and Crystalline State. Journal American Chemical Society, 2006. 128: p. 13454-13462.

2. R. Wu and T.B. McMahon, Infrared Multiple Photon Dissociation Spectra of Proline and Glycine Proton-Bound Homodimers. Evidence for Zwitterionic Structure. Journal American Chemical Society, 2007. 129: p. 4864-4865.

3. P. Zhang, et al., Neutron spectroscopic and Raman studies of interaction between water and proline. Chemical Physics, 2008. 345: p. 196-199.

4. M. Civera, M. Sironi, and S.L. Fornili, Unusual properties of aqueous solutions of L-proline:A molecular dynamics study. Chemical Physics Letters, 2005. 415: p.

274-278.

5. B. Schobert and H. Tschesche, Unusual solution properties of proline and its interaction with proteins. Biochimica et Biophysica Acta, 1978. 541: p. 270-277.

6. H.-D. Belitz, W. Grosch, and P. Schieberle, Food Chemistry 2009: Springer.

7. M. Jelifiska-Kazimierczuk and J. Szydlowski, Isotope Effect on the Solubility of Amino Acids in Water. Journal of Solution Chemistry,, 1996. 25(12): p. 1175-1184.

8. B.A. Wright and P.A. COLE, Preliminary examination of the crystal structure of l-proline. Aeta Cryst., 1949. 2: p. 129-130.

9. Y. Hayashi, et al., Large Nonlinear Effect Observed in the Enantiomeric Excess of Proline in Solution and That in the Solid State. Angew. Chem. Int. Ed., 2006. 45: p.

4593-4597.

10. S. Wurlitzer, et al., Micromanipulation of Langmuir-Monolayers with Optical Tweezers. J. Phys. Chem. B, 2001. 105: p. 182-187.

11. E.J.G. Peterman, F. Gittes, and C.F. Schmidt, Laser-Induced Heating in Optical Traps. Biophysical Journal, 2003. 84: p. 1308-1316.

12. Liu Y., et al., Physiological Monitoring of Optically Trapped Cells: Assessing the

Effects of Confinement by 1064-nm Laser Tweezers Using Microfluorometry.

Biophysical Journal, 1996. 71: p. 2158-2167.

13. S. Ito, et al., Application of Fluorescence Correlation Spectroscopy to the Measurement of Local Temperature in Solutions under Optical Trapping Condition.

J. Phys. Chem. B, 2007. 111: p. 2365-2371.

14. D. Walgraef, N.M. Ghoniem, and J. Lauzeral, Deformation pattern in thin films under uniform laser irradiation. Physical Review B, 1997. 15: p. 361-376.

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3. Laser Trapping Crystallization of L-proline

in Deuterated Water (D

2

O)

Based on the experience in glycine, laser trapping crystallization has only been achieved by focusing near-infrared laser to the air/solution interface [1]. It suggests that utilization of the interface increases possibility of crystallization. Thus we follow the previous example, L-proline crystallization was examined by setting the focus at the air/solution interface. Here I used bottom glass dish to prepare thin layered sample solution.

3.1 Surface deformation, crystallization, and dry spot formation

As mentioned in the previous chapter, local heating induced by irradiation is inevitable. Induced surface temperature elevation decreases the surface tension causes local solution surface deformation and height change along the distribution of Gaussian laser beam [2-5]. As we see in Fig. 3.1a solution surface height became lower by focusing trapping laser to the surface. Lowering rate depends on applied laser power. Higher power shows faster change of the height. It can be explained by higher temperature elevation with higher power. Interestingly, the lowering rate of solution height depends on the initial solution height as shown in Fig.3.1b. If initial solution height exceeds 100 m, it takes much longer time solution layer to be very thin. Because not only the walled container suppressed the solution

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deformation, but also high viscosity of proline solution resisted the surface change.

By further irradiation of trapping laser after lowering the surface height to be very thin (1~5 μm), crystallization was frequently observed. However, the crystal shape was always flat polycrystal and it grew quite rapidly probably due to its layer thinness and high concentration. Actually its size can reach up to more than 100 m within 1 sec. Fig. 3.2a shows a bright-field image of trapping-induced crystal which was taken by CCD camera

Fig. 3.1 (a) Power and (b) initial solution height dependent local solution height change. Supersaturated value was 0.9 SS. Power dependence measured with 0.5, 0.7, 0.9, 1.0 W and no laser irradiation. Lower panel shows initial solution height dependence measured with 30, 60, 80, 85 and 95 m. Laser power was 1.0 W.

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under microscope. Whole view of the crystal in the container is depicted in Fig. 3.3a. Solution height could not be checked anymore after crystallization, but it seemed that the height is slightly recovering.

Similar polycrystals can be formed when the solvent was evaporated. Crystals formed by self-evaporation are shown in Fig. 3.3b. We frequently observed that crystallization started from the outside of microscope view and propagation of the growth front passed whole view.

In contrast, we sometime observed a small dry spot at the surrounding of the laser spot as seen in Fig. 3.2b. No crystallization was observed in this case. Drying indicates absence of both solvent and solute molecules around the laser spot. It should be noted that observed dry spot formation is quite local phenomena occurring only near the focused laser spot, and surprisingly there is enough amount of the solution outside of the spot. We repeatedly observed that solution flowed back to the focal point when the laser irradiation was

Fig. 3.2 Further irradiation, solution height was decreased to near the bottom glass substrate and then gave two results. (a) Crystallization (b) Local totally dry, absence of solution.

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terminated. It obviously indicates that the dry spot is formed by surface lowering and touching the bottom dish surface.

3.2 Crystallization probability: Laser power and solution concentration

dependences

The probability of crystallization (forming the flat crystal) was examined by varying trapping laser power and solution concentration. All the measurements were done for 20 minutes and repeated at least 10 times for each condition. For concentration dependence experiments, sample solutions with lower supersaturated value were mainly employed because we could not avoid spontaneous crystallization which is occurred easily in highly saturated solution.

Fig. 3.4 shows results of concentration and trapping laser power dependence. Examined

(51)

concentration range is from 5.2 to 6.5 M which corresponds from 0.83 to 1.03 in supersaturated value. Supersaturated value is defined as the ratio of the weight of dissolved solute to that of the solute equals to 1.0 (1.9 g/mL for proline) [6]. Although we examined unsaturated to supersaturated condition, obtained results did not show obvious concentration dependence. Crystallization probability ranging from 40~65% indicates negligible concentration dependence.

On the contrary, crystallization probability showed obvious power dependence. It showed drastic probability change from 30% at 0.8 W to 90% at 1.2 W as shown in Fig. 3.3. We will discuss on this point more detail in chapter 3.4.

14/30 14/25 11/18 11/23 6/10 Pr oba bi lit y of c ry st all iz at io n (% ) Supersaturated value (a) 3/10 4/10 9/10 Prob abi lity of cry sta llizati on (%) (b) Laser power (W)

Fig. 3.4 (a) Concentration and (b) power dependence on crystallization probability of L-proline in D2O. Concentrations were 0.83, 0.88, 0.93 0.98 and 1.03 SS under fixed laser

power (1 W). Power dependence measured at 0.8, 1.0 and 1.2 W. Supersaturated value was 0.83 SS. Raw values of crystallization probability were mentioned at the bottom each bar.

脯氨酸雷射捕陷結晶化於溶液中之研究

國

立

交

通

大

學

應用化學系碩士班

碩

士 論 文

脯氨酸雷射捕陷結晶化於溶液中之研究

Laser Trapping Crystallization of L-proline in Solution

研 究 生：黃 重維 (Chong-Wei Huang)

指導教授：三浦 篤志 博士 (Dr. Atsushi Miura)

中 華 民 國 一 百 年 七 月

脯氨酸雷射捕陷結晶化於溶液中之研究

Laser trapping crystallization of L-proline in solution

研 究 生：黃重維 Student：Chong-Wei Huang

指導教授：三浦篤志 博士 Advisor：Dr. Atsushi Miura

國 立 交 通 大 學

應用化學系碩士班

碩 士 論 文

A Thesis

Submitted to M. S. Program

Department of Applied Chemistry

National Chiao Tung University

in Partial Fulfillment of the Requirements

for the Degree of

Master

in

Applied Chemistry

June 2011

Hsinchu, Taiwan, Republic of China

Laser Trapping Crystallization of L-proline in Solution

Student：Chong-Wei Huang Advisor：Dr. Atsushi Miura

M. S. Program, Department of Applied Chemistry

National Chiao Tung University

Abstract

脯氨酸雷射捕陷結晶化於溶液中之研究

中文摘要

Acknowledgement

Table of Contents

List of Figures

List of Tables

1. Introduction

1.1 Laser trapping

1.1.1 History

1.1.2 Principle of laser trapping

……… (1.2)

1.1.3 Laser trapping-induced assembly formation

1.1.4 Laser trapping crystallization

1.2 Crystallization

1.2.1 History and study of biomolecular crystallization

1.2.2 Crystallization theory

1.2.3 Laser-induced crystallization

1.2.3.1 Photochemical reaction induced crystallization

1.2.3.2 Optical crystallization

1.3 Motivation

1.4 References

2. Experimental

2.1 Materials

2.2 Microscope set up: Imaging and spectroscopy

2.3 Characteristics of proline

2.4 Estimation of local temperature elevation induced by trapping laser

irradiation



10

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10

e

NA

n

4

1

Z









士論文

研究生：黃重維 (Chong-Wei Huang)

指導教授：三浦篤志博士 (Dr. Atsushi Miura)

中華民國一百年七月

研究生：黃重維 Student：Chong-Wei Huang

指導教授：三浦篤志博士 Advisor：Dr. Atsushi Miura

國立交通大學

碩士論文

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