High-Throughput Crystallization of l

Subscriber access provided by UNIV OF YORK

Article

High-Throughput Crystallization of L-alanine using iCrystal Plates and Metal-Assisted and Microwave-Accelerated Evaporative Crystallization Muzaffer Mohammed, Yehnara S.B. Ettinoffe, Taiwo O Ogundolie, Bridgit M Kioko, Kevin Mauge-Lewis, and Kadir Aslan Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.5b04427 • Publication Date (Web): 10 Feb 2016 Downloaded from http://pubs.acs.org on February 17, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Industrial & Engineering Chemistry Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27

A

21 wells_5 cm

95 wells_10 cm

B

Silver (1 nm)

Nickel (10 nm)

Copper (10 nm)

Gold (1 nm)

204 wells_10 cm C

0.12

1 : Silver 2 : Nickel 3 : Copper 4 : Gold

0.10

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Industrial & Engineering Chemistry Research

Figure 1. A) Real‐color photographs of iCrystal plates with 21‐,95‐ and 204‐well sample capacity before the deposition of metal thin films B) Real‐color photographs of iCrystal plates with 21‐well sample capacity with different metal thin films C) UV‐vis absorbance spectrum of metal thin films on iCrystal plates.

0.08

3 1

0.06 2 0.04 0.02 0.00 400

ACS Paragon Plus Environment

4 450

500

550

600

Wavelength (nm)

650

700


Page 2 of 27

1 2 3 4 5 21‐Wells 95‐Wells 204‐Wells 6 PL 7 PMMA Ag 1 nm Ag 10 nm PMMA Ag 1 nm Ag 10 nm PMMA Ag 1 nm Ag 10 nm 8 RT_95 well_10 nm RT_21 well_PMMA 9 10 RT 11 12 395 330 265 140 280 95 200 100 120 13 14 15 1 16 17 65 115 40 95 60 65 50 55 84 PL1_21 well_10 nm 18 PL1_204 well_10 nm 19 20 3 21 22 20 15 20 20 30 30 20 18 16 23 24 25 5 26 27 PL10_21 well_10 nm PL5_204 well_1 nm 15 9 18 15 8 10 8 8 16 28 29 30 7 31 32 14 6 16 13 6 9 6 6 16 33 34 35 10 36 37 8 4 8 11 4 8 4 4 4 38 Figure 2. Optical microscope images of L‐alanine crystals on iCrystal plates with 21‐, 95‐ and 204‐well sample capacity with 1 nm Ag and 10 nm silver films 39 and without silver thin film (blank PMMA) at room temperature and using microwave (MW) heating. (Inset: Complete crystallization time in minutes). PL: 40 41 Microwave Power Level. Scaler bar = 500 m. 42 43 44 45 46 ACS Paragon Plus Environment 47 48

Page 3 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


RT_PMMA_cover

Optical Images of Alanine Crystals / Complete Evaporation (Time, min)

21-wells Blank PL

10 nm Ag

1 nm Ag

PMMA with cover

With cover only

With well‐coat only

With cover with well‐coat

With cover only

With well‐coat only

With cover with well‐coat

RT

300

170

180

300

235

290

300

1

170

105

50

125

55

80

125

3

75

70

20

35

45

35

14

5

20

16

15

19

18

7

8

PL10_1 nm_coat/cover 7

11

6

4

7

3

9

8

10

8

5

3

5

1

3

4

PL1_1 nm_well coat

PL5_1 nm_cover

Figure 3. Optical microscope images of L‐alanine crystals formed on iCrystal plates with silver thin film and without silver thin film (blank PMMA) and at room temperature and using microwave (MW) heating. A polymer lid for covering was used to cover the wells. PL: Microwave Power Level. Scaler bar = 500 m.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Optical images of L‐Alanine Crystals Complete Evaporation Time, min

21-wells MW_Ni_90 C

Initial Temp

MW_Au_90 C

Silver 1 nm (429 W/mK) RT

Page 4 of 27

MW

Copper 10 nm (401 W/mK) RT

MW

Gold 1 nm (310 W/mK) RT

MW

Nickel 10 nm (91 W/mK) RT

RT_Ni_50 C

MW

50°C

RT_Au_50 C 120

55

325

55

145

60

270

55

195

55

115

55

155

65

305

65

215

60

175

90

230

55

275

60

70°C MW_Cu_90 C

RT_Cu_50 C

90°C

Figure 4. Summary of L‐alanine crystallization on iCrystal plates with 21‐well sample capacity with various metal thin films and without a polymer cover at room temperature and using microwave (MW) heating. Initial temperature of the solutions used were 50°C, 70°C and 90°C. Scaler bar = 500 m.


Page 5 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


95‐Wells

Power Level

PMMA

Ag 1 nm

Ag 10 nm

N/A (RT)

251‐1405 45

140‐1554 158

71‐1519 280

1

75‐1427 294

139‐1429 128

95‐1414 386

3

130‐1795 263

106‐733 126

270‐541 4

5

120‐1800 158

47‐295 85

125‐921 5

7

112‐1832 247

62‐247 1

89‐326 4

10

113‐1815 260

66‐136 4

147‐431 12

PMMA_PL 1

1 nm Ag_PL 1

10 nm Ag_PL 1

Size Range, µm Number of Crystals

Figure 5. Summary of L‐alanine crystallization using iCrystal plates with 95‐well sample capacity at room temperature and using microwave (MW) heating. PMMA contains no silver thin films. Scaler bar = 500 m



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

204‐Wells

PMMA_PL 1

Page 6 of 27

Size Range, µm Number of Crystals

Power Level

PMMA

Ag 1 nm

Ag 10 nm

N/A (RT)

90‐1248 78

55‐1524 30

76‐847 91

1

92‐1130 29

59‐625 154

66‐1107 97

3

96‐364 25

90‐257 54

101‐1481 38

5

103‐240 4

171‐282 5

105‐1533 63

7

126‐355 17

N/A

220‐2665 41

10

N/A

N/A

155‐1718 59

1 nm Ag_PL 1

10 nm Ag_PL 1

Figure 6. Summary of L‐alanine crystallization using iCrystal plates with 204‐well sample capacity at room temperature and using microwave (MW) heating. PMMA contains no silver thin films. Scaler bar = 500 m


Page 7 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


High-Throughput Crystallization of L-alanine using iCrystal Plates and MetalAssisted and Microwave-Accelerated Evaporative Crystallization Muzaffer Mohammed, Yehnara S.B. Ettinoffe, Taiwo O. Ogundolie, Bridgit M. Kioko, Kevin Mauge-Lewis and Kadir Aslan* Morgan State University, Department of Chemistry, 1700 East Cold Spring Lane, Baltimore, MD 21251 USA *Corresponding Author: [email protected] ABSTRACT We present a comprehensive study of high-throughput crystallization of L-alanine (a model amino acid) using circular crystallization platforms (are hereafter referred to as the iCrystal plates) designed to work with the Metal-Assisted and MicrowaveAccelerated Evaporative Crystallization (MA-MAEC) technique. The iCrystal plates are constructed using a circular 21-, 95- and 204-well design that afford for homogenous microwave heating of all samples. In addition, the iCrystal plates were modified with metal thin films (gold, copper, silver and nickel), which act as selective nucleation sites for selective crystallization to occur on the surface of the crystallization platforms. Silver thin films were found to be ideal for MA-MAEC applications as compared to other metal surfaces based on the observations of crystal number and size. The size and number of wells on the iCrystal plates were found to have negligible effect on the growth of Lalanine crystals, where all three iCrystal plates were found to have similar crystallization times and yield identical crystal quality. These results imply that one can employ the iCrystal plates for the crystallization of small number of samples (with 21-well capacity)

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 27

and large number of samples (95- and 204-well capacity) with identical yields using the MA-MAEC technique. Keywords: Plasmonic thin films, rapid crystallization, crystallization platforms, l-alanine, amino acids. INTRODUCTION Crystallization is essential to chemical, biotechnology and pharmaceutical industries, as a link between a newly synthesized or isolated compounds and their subsequent characterization studies. However, crystallization of macromolecular compounds can take days or sometimes up to weeks; therefore a rapid crystallization technique is required that can expedite crystallization without affecting the quality of crystals. For example, new drug entities (NDEs) are designed based on Quantitative Structure Activity Relationship (QSAR) models, a process that reduces the overall drug development time by identifying the molecular designs most identical and compatible with the target biological receptor.1-7 Crystallization is used to identify the morphology, habit and lattice energy of a crystal and to predict its structure-activity relationship (SAR).8-10 Information related to the structure, functions and behavior of NDEs under various conditions are not well-established. Crystallization of such entities on a laboratory scale and on-demand basis can expedite their characterization and bioactivity studies, resulting in enormous savings in terms of time, labor and money.11-13 The field of plasmonic materials has gained notable interest due to their thermal conductive abilities that can be utilized in a wide range of applications.14-17 Plasmonic materials also have coherent oscillations of conduction electrons due the interactions of light with the metal dielectric environment. 14, 15, 18-22 Some plasmonic materials, such as 2 ACS Paragon Plus Environment

Page 9 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


silver, copper, gold and nickel have been utilized in numerous applications in biotechnology.23-28 For example, silver and gold have optical properties that are useful in as diagnostic devices due to the distinct bright colors they display during plasmon resonance.23,

29-35

Nickel nanostructures which exhibit optical and magnetic properties

are being used for data storage and in various sensors.36-39 In particular, nickel is suitable for enhancing near-infrared fluorescence because of its electric field has a broad range (500 nm-800 nm).36 Copper nanostructures have been used for medical application such as glucose sensing due to its high electro-catalytic properties.40,

41

These metals are relatively inexpensive, non-toxic, easily manufactured and can be stored for a longer period of time that makes them attractive options for such applications.42 Our research laboratory has recently introduced and demonstrated the applications of a crystallization technique, called MA-MAEC.14,

43-45

In the MA-MAEC

technique, plasmonic materials are utilized as nanoparticle or thin films deposited on a circular multi-well poly(methyl methacrylate) (PMMA) platform for the crystallization of compounds in a rapid manner. The effectiveness of the MA-MAEC technique in rapid crystallization is attributed to the phenomenon called Microwave Induced Thermal Gradient (MITG), which describes a thermal gradient formed between the metal thin films and the solution upon exposure to microwave radiation.36,

42, 46-48

The metal thin

films have a significantly higher thermal conductivity (e.g., ksilver = 429 W/mK), in comparison to water (0.58 W/mK), that results in the formation of thermal gradient driving the molecules towards cooler metal surface that serve as a selective nucleation site for the molecule to bind and subsequently accelerating the crystal formation.19 We



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 27

have previously demonstrated the existence of MITG between the bulk and the metal surface by theoretical calculations and real-time experimental measurements of the temperatures of the bulk and the metal surfaces.46, 49, 50 The reader is referred to these publications for further details of the MITG phenomenon. Since MITG exists between the bulk (warmer) and the metal surface (cooler), the direction of mass transfer can be predicted. In this comprehensive study, we show the application of the MA-MAEC technique for high throughput crystallization of a model molecule (L-alanine) on our three different iCrystal plates: 1) with small number of samples (21-well), 2) medium scale (95-well) and 3) large scale (204-well) output. Crystallization of L-alanine was carried out at room temperature and using different microwave heating power levels (PL 1,3,5,7 and 10), in a conventional domestic microwave oven. The variation of microwave power level, which is related to duty cycle of a microwave source, was investigated to study the effect of MITG in the crystallization of L-alanine. In addition, the following parameters: 1) plasmonic materials (silver, gold, copper and nickel thin films), 2) thickness of plasmonic material thin film (1 nm and 10 nm) and 3) initial temperature of L-alanine solution were investigated. Our results show that microwave heating at PL 1 for 1 nm silver thin film platform results in larger L-alanine crystals in comparison to all other experimental conditions. We also observed that use of silver thin films on the iCrystal plates produces L-alanine crystals of higher quality in terms of size, number and crystal morphology, as compared to gold, copper and nickel thin films. The increase in the initial solution temperature was found to yield smaller crystals and rapid solution evaporation. All three iCrystal plates were found to yield L-alanine crystals similar in size and number in all


Page 11 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


wells. These observations can be attributed to the unique well pattern of the iCrystal plates that distributes microwave radiation in a homogenous fashion across all wells, which affords for the rapid crystallization time and consistent crystal quality. MATERIALS AND METHODS Materials. L-alanine was purchased from Sigma-Aldrich, USA.190-proof ethanol (95%) was purchased from Pharmco Products Inc. Deionized water (DI water) purified via Millipore Direct Q3 UV apparatus (>18.0 MΩ.cm resistivity at 25°C). EMS 150RS sputtering device, silicone isolators with 21-, 95- and 204 well capacity (volume = 30 µL) and metal targets (silver, copper, gold, and nickel; 57 mm in diameter) were purchased from Electron Microscopy Sciences, PA, USA. PMMA discs (5 and 10 cm in diameter) and polymer cover were purchased from McMaster-Carr, IL, USA. A 900 W Emerson microwave oven (Model no. 0009443) was purchased from Walmart, Inc., USA. Cary 50 Bio UV-visible spectrophotometer was purchased from Varian, USA. Harrick PDC-32G plasma cleaner was purchased from Harrick’s Inc., NY, USA. Methods Preparation of the Solution. Solution of L-alanine were prepared in glass vials with 2.4 g of L-alanine in 10 mL of DI water until a clear solution was obtained. L-alanine solutions were maintained at various initial temperatures of 50°C, 70°C, and 90°C for temperature vs crystallization time studies. Preparation of iCrystal Plates. Blank PMMA discs (5 cm in diameter for 21-well design and 10 cm for 95-well and 204-well designs) were cleaned using ethanol (190 proof) and DI water, and air dried. Plasmonic thin film layers were deposited under argon environment at selected thickness levels and the deposition was monitored using 5 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 27

an in-built thickness monitor equipped with a quartz crystal microbalance. A silicon isolator either with 21-, 95- or 204-wells were attached on the metal-deposited PMMA discs. The name iCrystal plates refers to those PMMA discs with a silicon isolator (Figure 1A). The design and theoretical heating pattern studies of the iCrystal plates have been published previously by our laboratory.14,

44, 51

Figure 1B shows optical

images of 21-well iCrystal plates with silver (1 and 10 nm), gold (1 nm), nickel (10 nm) and copper (10 nm) thin films used in this study. In the current design, each well of the iCrystal plates with 21-wells and 95-wells can hold up to 30 µL solution, while each well of the iCrystal plates with 204 well can hold up to 10 µL solution. One can increase the capacity of the wells by affixing additional silicon isolators (not employed in this study). Characterization of Plasmonic Thin Films on iCrystal Plates. The UV-vis absorbance spectrum (400 nm-700 nm) was used to characterize plasmonic thin films deposition on iCrystal plates. Figure 1C confirms that the plasmonic thin films were deposited as thin films. It is important to note the dielectric constants of these metals and their surface plasmon resonance wavelengths differ significantly, and subsequently, the color of the thin films, which depends on the thickness of the films, also is different from another. Crystallization of L-Alanine Crystals. A 20 µL of L-alanine solution for iCrystal plates with 21-wells and 95-wells or 10 µL for 204-well iCrystal plates was dispensed manually (using a Viaflo 12-channel micropipette, catalog no: 4132) into each well and observed under an optical microscope for crystal growth at room temperature. In case of MA-MAEC technique, after dispensing the solution, the iCrystal plate was subjected to microwave heating at different power levels i.e. 1,3,5,7 and 10.14, 44 Please note that


Page 13 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the microwave power levels in the 900 W microwave oven used in this study is related to the duty cycle of the microwave oven. That is, for a total microwave heating time of 5 minutes at microwave power level 1, full 900 W power is on for 3 sec and is automatically turned off for 27 sec until the set time is reached. Detailed explanation of these observations can be found in our earlier publications.14,

43-45

After each heating

cycle of 5 min, the iCrystal plates were removed from the microwave oven and optical images of the crystals were taken using an optical microscope. Crystallization time reported is based on complete evaporation of the solution from the well, unless stated otherwise. Characterization

of

L-Alanine

crystals.

Crystals

of

L-alanine

were

characterized using optical microscopy. Crystals were also observed for their length, number of crystals per well and initial crystal appearance time using an optical microscope (Swift Digital M10L monocular microscope was connected to a PC equipped with an image processing software provided by the vendor). RESULTS AND DISCUSSION The effect of metal thickness on the high-throughput crystallization of L-alanine Since the MA-MAEC technique is based on the use of metal films in conjunction with microwave heating, the type and physical properties of the metal films are expected to play a significant role for high-throughput crystallization of molecules. In this regard, based on our previous observations with silver nanoparticulate films for low throughput crystallization of molecules,14, 44 we first employed silver metal for the high-throughput crystallization studies. Two different thickness of silver thin films (1 nm and 10 nm) were used in order to investigate whether the thickness of the metal film can affect the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 27

crystallization of L-alanine: while 1 nm silver films are semi-continuous (i.e., nanoparticulate) surface, 10 nm silver film are continuous with relatively smoother surface. Semi-continuous silver film offer preferential nucleation sites for L-alanine molecules due to the chemical interactions between the amine groups of the L-alanine molecule with silver, as compared to unsilvered polymer surface. The nucleation of Lalanine crystal can occur at any part of the continuous silver films and still take advantage of amine-silver chemical interactions. Figure 2 shows the summary of observations for L-alanine crystals grown at room temperature (a control experiment, no microwave heating) and using the MAMAEC technique (power levels 1,3,5,7 and 10) on three different iCrystal plates with 21, 95- and 204-well capacity. In addition, to demonstrate the advantage of using metal films in the MA-MAEC technique, a second control experiment, where the silver film was omitted from the iCrystal plates (labeled as PMMA or blank iCrystal plates) was carried out. For example, at room temperature, the time taken for complete evaporation of solution on iCrystal plates with 21-well sample capacity was 100 min, 120 min and 140 min for the blank, with 1 nm silver film and 10 nm silver film, respectively. In addition, at room temperature, the complete evaporation time was increased as the sample capacity of the iCrystal plates is increased. Using MA-MAEC, the complete evaporation of identical solution on iCrystal plates with 21-well sample capacity occurred as low as 4 min for power level 10. Similar reduction in the time for complete evaporation time on iCrystal plates with 95-wells and 204-wells was observed using the MA-MAEC technique (Figure 2). These results show that the time for complete evaporation of solvent can be reduced over ~25-fold for the blank iCrystal plates and 30-fold for


Page 15 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


iCrystal plates with 1 nm and 10 nm silver thin films and the thickness of the silver metal film has no significant effect on the high-throughput crystallization of L-alanine using the MA-MAEC technique. Subsequently, one can choose any thickness of a metal film on the iCrystal plates for MA-MAEC based crystallization of target molecules. Although the total time for crystallization for all iCrystal plates after microwave heating are similar, it is important to compare the differences in the size and shape of Lalanine crystals grown on different surface conditions (Figure 2 and S1, these images were taken after complete evaporation of solution). At room temperature, L-alanine crystals appeared to be smaller, spread out on the surface and fewer in numbers. However, the use of MA-MAEC technique resulted in an increase in the number and size of crystals per well as the power level of microwave was increased. The use of microwave power level 10 (i.e., 900 W is on 100% of the set time) resulted in the rapid growth of L-alanine crystals, which are joined in several crystal faces, as compared to other L-alanine crystals grown using microwave power level 1 to 7, where individual crystals can be observed. The wells of the iCrystal plates were kept open to allow complete evaporation of the solvent in all experiments described to this point. These observations imply that one can control the L-alanine crystal shape and size by varying the microwave power level in a conventional domestic microwave oven and using iCrystal plates without a cover to allow rapid evaporation of the solvent. The effect of design of iCrystal plates on the high-throughput crystallization of Lalanine In this section, two additional parameters for the high-throughput crystallization of L-alanine were investigated: 1) the use of a polymer cover to control the evaporation of



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 27

solvent and 2) metal coating of the wells for improved crystallization. The complete evaporation of the solvent is desirable in certain crystallization applications, such as in the crystallization of organic and inorganic compounds, however, the evaporation of the solvent is required to be slower and/or avoided in other applications. In addition, the deposition of metal thin films on to iCrystal plates occur only on the PMMA surface and the silicon isolators are added on to the PMMA surface after the deposition of the metal thin film. Subsequently, L-alanine solution are exposed to polymer surface with metal film coating and the surface of the silicon isolator without metal thin films, which creates a heterogeneous surface for the growth of L-alanine crystals and can play in an important role in the crystal growth. To investigate the role of these two parameters, a polymer cover was used to seal the wells as a means to control the evaporation of the solution and the 21-well iCrystal plate with pre-attached silicone isolator was used as a target to achieve uniform thin film deposition including the walls of the wells. Several crystallization experiments were carried out using 21-well iCrystal plates with silver thin film: a) without silver metal film and with polymer cover (control experiment) b) with polymer cover only c) with well-coat only and d) with polymer cover and well-coat. Figure 3 shows crystallization times and representative optical images of Lalanine crystals grown using the polymer cover and well coating of the iCrystal plates with silver (all experiments were repeated at least five times to ensure repeatability of the experiments described in this study, data not shown for the sake of brevity). It was observed that both well-coat and polymer cover has a significant effect on the evaporation time, crystal number and size. The iCrystal plate with 10 nm silver thin film with polymer cover only and microwave heating at power level 10 resulted in the fastest


Page 17 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


crystallization time (~4 min), however the L-alanine crystals were small in size and fewer in number. With the use of iCrystal plates well-coat only and microwave heating, the crystal size was increased in comparison to crystals grown on iCrystal plates without well-coat. The growth of larger L-alanine crystals resulted in the reduction of the total number of crystals obtained per well. Using iCrystal plates with both the well-coat and the polymer cover with microwave heating: 1) crystallization times were reduced, 2) crystal size was increased and 3) the number of crystals per well was lower as compared to other conditions. More importantly, using iCrystal plates with only wellcover on both 1 nm and 10 nm silver platforms with microwave heating (PL 1 and 5), one can grow larger L-alanine crystals in size and number as compared to all other conditions (Figure S2). These observations demonstrate that in order to achieve consistent crystal quality using the iCrystal plates, a polymer cover should be used with the iCrystal plates to control the evaporation rate of the solution and well-coating of the iCrystal plates with metal film is necessary to avoid exposure of the crystallization medium to heterogeneous surfaces for improved crystallization of L-alanine in a high throughput manner. Figure S2 also shows that the number of L-alanine crystals is decreased in the experimental conditions shown as the microwave power level is increased, which can be attributed to higher evaporation rates due to the increase in microwave power level that results in higher nucleation rates. However, no significant changes in the size range of the L-alanine crystals were observed, which is yet to be fully understood. The effect of type of metal film and initial solution temperature on the highthroughput crystallization of L-alanine



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 27

To date, the application of MA-MAEC technique to rapid crystallization of target molecules was only demonstrated using silver metal in nanoparticle and in thin film form. In this regard, we investigated the use of three new metal thin films (gold, copper and nickel) in addition to silver on iCrystal plates with 21-well sample capacity without a polymer cover. It should be noted that the thickness of each metal thin films is different due to their stability and ease of deposition on the iCrystal plates and do not play a significant role in the high-throughput crystallization of L-alanine (Figure 1). In addition, crystallization experiments were performed with three L-alanine solutions prepared at different initial temperatures: 50°C, 70°C and 90°C, to study the effect of higher initial temperature on the crystallization of L-alanine for the following reason: the crux of the MA-MAEC technique is the maintenance of largest possible extent of MITG between the solvent and the metal surface to accelerate the mass transfer of molecules from warmer solvent to the cooler metal surface while the solvent is evaporated. In this regard, the effect of the initial temperature of L-alanine solution on the crystallization of L-alanine (in terms of complete evaporation time, number of crystals grown) was investigated to study whether the evaporation of solvent can be accelerated while maintaining the largest possible extent of MITG as the initial temperature of the L-alanine solution is increased. Figure 4 shows optical images and complete evaporation times of the L-alanine solution at different initial temperatures using iCrystal plates with 21-well sample capacity and four different metal thin films. The fastest time for crystallization was observed at ~55 min using microwave heating at power level 1 for all metal surfaces, whereas the longest time of ~275 min was observed at room temperature on iCrystal


Page 19 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


plates with nickel thin film. The initial solution temperature appeared to have an effect on complete evaporation time, where an increase in complete evaporation time was observed with L-alanine solution at 90°C in comparison to solution at 50°C for all surfaces except nickel, which had a longer time for 70°C solution. L-alanine crystals grown on gold, copper and nickel thin film surfaces were on average smaller in size as compared to those grown on silver surface (Figure S3). The use of iCrystal plates with silver thin film and L-alanine solution at 50°C along with microwave heating resulted in the growth of the most number of L-alanine crystals (~154) and ranging in size between 59-625 µm (Figure S3). The observed differences in the complete solvent evaporation time can be attributed to the differences in the thermal conductivities of the metal surfaces, which results in different rate of cooling of L-alanine solution. These results imply that one can employ copper, gold and nickel thin films on iCrystal plates, especially when the cost and availability of these metals are the main concern of the research and development laboratories. The effect of sample capacity on the high-throughput crystallization of L-alanine As mentioned earlier in the text, iCrystal plates also offer a high throughput designs with 95- with 204-well sample capacity manufactured using a circular PMMA disc 10 cm in diameter. Figure 5 shows summary of L-alanine crystallization data obtained on iCrystal plates with 95-well sample capacity (blank, 1 nm and 10 nm silver thin film) at room temperature and using microwave heating. L-alanine crystals grown on 10 nm silver thin films using microwave heating at power level 1 were as large as ~1,200 µm in size and quantity (total number of L-alanine crystals grown on all 95 wells = 386). Application of microwave heating at power level 3 to 10 on iCrystal plates with



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 27

silver thin films resulted in rapid evaporation of solution, thereby significantly reducing the size and quantity of L-alanine crystals grown. At room temperature (i.e., no microwave heating), total time taken for complete evaporation of the solvent was >300 min, whereas using microwave heating the complete evaporation was achieved in less than ~70 min for power level 1 (Figure S4), ~10 min for power level 5 (Figure S5) and ~8 min for power level 10 (Figure S6). Figures S4, S5 and S6 also show optical images of iCrystal plates with 95-well sample capacity after complete crystallization using microwave heating at power level 1, 5 and 10. As observed in these images, all wells on the iCrystal plates show L-alanine crystals were grown simultaneously. Similar results were obtained on iCrystal plates with 204well sample capacity (Figure 6), where iCrystal plates with 10 nm silver and using microwave heating at power level 1 yielded the largest number of L-alanine crystals (~97) with sizes ranging between 66-1107 µm. It should be noted that despite the large size of iCrystal plates with 95- and 204-well sample capacity (i.e., 10 cm), the evaporation of the solution from all wells was observed to be similar; which is attributed to the homogenous heating obtained with the novel platform design. Data presented in this paper conclusively demonstrates the successful application of the MA-MAEC technique for rapid crystallization of L-alanine (a model amino acid) in a high throughput fashion, across wide spectrum of conditions that are encountered by chemists engaged in crystallization research. More specifically, this paper yielded new knowledge as listed in the following: a) The suitability of different metal surfaces for selective nucleation and crystallization of biological molecules,


Page 21 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


b) Optimum microwave heating time and power level for a conventional microwave oven, c) Impact of initial solution temperature on crystallization, d) Metal thin film thickness and its impact on crystallization time, e) Relationship between microwave heating power and scale up factor for high throughput crystallization using MA-MAEC technique, and f) Sample size and output relationship. This study also highlights the significant feature of iCrystal plates, which is the scale up process from small scale platforms (21-well sample capacity) to medium laboratory scale (95-well sample capacity) and R&D scale (204-well sample capacity) platforms. One can use iCrystal plates during several stages of crystallization research, which includes initial crystallization screening, optimization of crystallization conditions and simultaneous crystallization of multiple target molecules. Our laboratory is currently working on the crystallization of peptides, and these results will be published in due course. Conclusions The successful application of MA-MAEC technique for rapid crystallization of Lalanine in a high throughput fashion using three different crystallization platforms (i.e., iCrystal plates) is demonstrated. Optical images revealed that L-alanine crystals grown on the all three iCrystal plates were well-defined, consistent and larger compared to Lalanine crystals grown at room temperature. Application of microwave heating significantly reduced the overall crystallization time for samples (each well contain 10-30 µL volume) from >300 min to

High-Throughput Crystallization of l

Recommend Documents