Thermo-Physical Properties, Binary Phase Diagram of Enantiomers

Mobeen Ashraf

2015

INVESTIGATING MIXTURES OF ENANTIOMERIC SOLVENTS: THERMO-PHYSICAL

PROPERTIES, BINARY PHASE DIAGRAM OF ENANTIOMERS, OPTICAL PURITY AND

SOLVENT PARAMETERS OF ETHYL LACTATE ENANTIOMERS

Mobeen Ashraf

ABSTRACT

A polar-protic enantiomeric solvent, ethyl lactate, is a green solvent because of its non- carcinogenetic, low toxicity, non-corrosivity, 100% biodegradability, high boiling point, and ease of recycling properties. The two stable enantiomers of ethyl lactate are (-)-Ethyl L-Lactate and

(+)-Ethyl D-Lactate. Ethyl Lactate is typically synthesized from ethanol and L-Lactic Acid because the D-form is more toxic. However a review of the literature indicated that certain thermo-physical properties such as density, specific optical rotation, specific gravity, refractive index, melting point, and binary phase-diagram of ethyl lactate enantiomers are either scarcely documented or missing altogether. In addition, the MSDS documented melting point of (-)-Ethyl

L-Lactate is incorrectly reported. There is very limited to no information on basic physical and chemical properties of (+)-Ethyl D-Lactate in literature such as melting point, refractive index, specific gravity, acidity. No binary phase diagram of ethyl lactate enantiomers have been constructed to distinguish between the melting temperatures of L-form, D-form and racemic mixture of ethyl lactate. Phase behavior and thermo-physical properties of solvents are important in developing an environmental friendly process. Therefore, an investigation was made to measure the melting point (Tm), density (ρ), refractive index (nD), specific optical rotation [α] and optical purity (% enantiomeric excess) of ethyl lactate enantiomers and their mixtures. Binary phase diagram of ethyl lactate enantiomers was constructed to determine the eutectic point in the D/L composition using modulated Differential Scanning Calorimetery (DSC).

Binary phase diagram of ethyl lactate enantiomers showed an eutectic point temperature of -

26.95°C at 60:40 [(L-form)/(D-form] composition of Ethyl Lactate. DSC results further determined that instead of documenting the melting point of (-)-Ethyl L-Lactate, manufacturers reported the melting point of ethyl lactate’s racemic mixture on the MSDS. The Tm of (-)-Ethyl L-

Lactate was -4.23 °C. The D-form has Tm of -4.23 °C and racemate has Tm of -25.64 °C. Optical rotation measurements coupled with GC-MS measurements indicated an optically active impurity in (+)-Ethyl D-Lactate, leading to higher optical activities than the (-)-Ethyl L-Lactate form.

Three solvents: (-)-Ethyl L-Lactate, (+)-Ethyl D-Lactate and their racemic mixture were further characterized using solvatochromic techniques. Polarizability/depolarization (π*), hydrogen-bond donating-acidity (α), and hydrogen-bond accepting-basicity (β) of (-)-Ethyl L-

Lactate, (+)-Ethyl D-Lactate and racemic mixture were investigated by using UV-Vis spectroscopy over a temperature range of T = (283.15 to 343.15) K. Reichardt’s parameter,

ET(30), was also investigated for (-)-Ethyl L-Lactate, (+)-Ethyl D-Lactate and racemic mixture under the same temperature conditions. Reichardt’s betaine dye, N,N-diethyl-4-nitroaniline and

4-nitroaniline were the three solvatochromic probes used to measure the ET(30) scales and the

Kamlet-Taft Parameters (α, β, and π*).

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ACKNOWLEDGEMENTS

This thesis would not have been possible with the constant support, guidance, patience and encouragement from my adviser Dr. Douglas Fox that he provided throughout the entirety of my project. Amidst of his busy schedule, he was always available to assist me during the most difficult times when writing this thesis.

I would also like to thank Dr. Shoaleh Dehghan, who assisted me incorporating Gas-

Chromatography Mass Spectrometery (GC-MS) measurements in this work. I thank my fellow lab mate, Solomon Teklai, who has been very supportive and confident on my abilities throughout my graduate studies. I would also like to thank Chemistry Faculty staff at US Naval

Academy in Annapolis, MD who gave me a tremendous opportunity to use their instrumentations. Last but not the least, I would thank all of the committee members for their time, valuable comments and guidance.

TABLE OF CONTENTS ABSTRACT ...... ii ACKNOWLEDGEMENTS ...... iv TABLE OF CONTENTS ...... v LIST OF ILLUSTRATIONS...... vi LIST OF TABLES ...... vii INTRODUCTION ...... 1 Objective ...... 5 Optical Rotation [α] ...... 6 Density (ρ) ...... 9

Refractive Index (nD) ...... 10 Differential Scanning Calorimetry (DSC) ...... 11 Solvatochromism ...... 16 Gas-Chromatography-Mass Spectrometry (GC-MS) ...... 24 EXPERIMENTAL SECTION...... 25 RESULTS AND DISCUSSION ...... 29 Optical Rotation [α] and Density (ρ) ...... 29

Refractive Index (nD) ...... 32 Differential Scanning Calorimetry (DSC) ...... 33 Solvatochromism ...... 36 Gas Chromatography-Mass Spectrometry (GC-MS) ...... 31 CONCLUSIONS ...... 43 APPENDIX A: GC-MS RESULTS ...... 45 APPENDIX B: MODULATED DSC THERMOGRAMS ...... 51 APPENDIX C: ABSORPTION SPECTRA ...... 56 REFERENCES ...... 62

LIST OF ILLUSTRATIONS Figure 1. The Molecular Structure of Ethyl Lactate ...... 2 2. Two Enantiomeric forms of Ethyl Lactate ...... 3 3. Schematic Diagram of a Polarimeter ...... 7 4. Binary Phase Diagram of Enantiomers showing the Eutectic Point ...... 13 5. DSC Thermogram showing Various Thermal Events ...... 14 6. Structures of Solvatochromic Probes used in this Study to Investigate Solvent Polarity and Hydrogen-Bonding Characteristics ...... 17 7. Molecular Structure of the Zwitterionic Reichardt’s Betaine Dye showing its Inherent Properties ...... 19 8. Molecular Structure of Zwitterionic Dichlorosubstituted Reichardt’s Betaine Dye ...... 20 9. Resonance Forms of 4-Nitroaniline ...... 21 10. HBA Sites in 4-Nitroaniline ...... 21 11. HBA Sites in N,N-Diethyl-4-Nitroaniline ...... 22 12. Binary Phase Diagram of Ethyl Lactate Enantiomers ...... 33

13. Plot Showing Temperature Dependence of ET(30) Polarity Scale in (-)-Ethyl L- Lactate, (+)-Ethyl D-Lactate and Racemic Ethyl-D,L-Lactate Mixture ...... 36 14. Plot Showing Temperature Dependence of HBA (β) for (-)-Ethyl L-Lactate, (+)- Ethyl D-Lactate and Racemic Ethyl-D,L-Lactate Mixture ...... 38 15. Plot Showing Temperature Dependence of HBD (α) for (-)-Ethyl L-Lactate, (+)- Ethyl D-Lactate and Racemic Ethyl-D,L-Lactate Mixture ...... 39 16. Plot showing Temperature Dependence of Polarizability (π*) for (-)-Ethyl L- Lactate, (+)-Ethyl D-Lactate and Racemic Ethyl-D,L-Lactate Mixture ...... 41 17. GC Chromatogram of (-)-Ethyl L-Lactate ...... 45 18. GC Chromatogram of (+)-Ethyl D-Lactate ...... 46 19. GC Chromatogram of Racemic Ethyl Lactate Mixture ...... 46 20. Mass Spectrum of the Main Peak in all Ethyl Lactate Solvents ...... 47 21. Mass Spectrum of the 2nd peak in (+)-Ethyl-D-Lactate ...... 47 22. Mass Spectrum of the 3rd peak in (+)-Ethyl-D-Lactate ...... 48 23. GC-MS Summary displaying the Percent Purity of (-)-Ethyl L-Lactate ...... 48 24. GC-MS Summary displaying the Percent Purity of (+)-Ethyl D-Lactate ...... 49 25. GC-MS Summary Displaying the Percent Purity of Racemic Ethyl Lactate ...... 50

26. Showing DSC Cooling Curves for Ethyl Lactate Enantiomeric Mixtures Obtained at a Heating Rate of 20 oC/min ...... 50 27. DSC Thermogram showing the Melting Point and Enthalpy of Fusion of (-)-Ethyl L-Lactate Solvent ...... 52 28. DSC Thermogram showing the Melting Point and Enthalpy of Fusion of (+)-Ethyl D-Lactate Solvent ...... 53 29. DSC Thermogram showing the Melting Point and Enthalpy of Fusion of Racemic Ethyl Lactate Solvent ...... 54 30. DSC Thermogram showing the Melting Point and Enthalpy of Fusion at Eutectic Point Composition: 60:40 [(L-form)/(D-form] of Ethyl Lactate Solvent ...... 55 31. Electronic Absorption Spectrum of Reichardt’s dye in (-)-Ethyl L-Lactate at T= 10, 25, 40, 55 and 70 oC ...... 56 32. Electronic Absorption Spectrum of Reichardt’s dye in (+)-Ethyl D-Lactate at T= 10, 25, 40, 55 and 70 oC ...... 56 33. Electronic Absorption Spectrum of Reichardt’s Dye in Racemic Ethyl Lactate Mixture at T= 10, 25, 40, 55 and 70 oC ...... 57 34. Electronic Absorption Spectrum of DENA dye in (-)-Ethyl L-lactate at T= 10, 25, 40, 55 and 70 oC ...... 57 35. Electronic Absorption Spectrum of DENA dye in (+)-Ethyl D-lactate at T= 10, 25, 40, 55 and 70 oC ...... 58 36. Electronic Absorption Spectrum of DENA Dye in Racemic Ethyl Lactate Mixture at T= 10, 25, 40, 55 and 70 oC ...... 58 37. Electronic Absorption Spectrum of PNA dye in (-)-Ethyl L-lactate Solvent at T= 10, 25, 40, 55 and 70 oC ...... 59 38. Electronic Absorption Spectrum of PNA dye in (+)-Ethyl D-Lactate at T= 10, 25, 40, 55 and 70 oC ...... 59 39. Electronic Absorption Spectrum of PNA dye in Racemic Ethyl Lactate Mixture at T= 10, 25, 40, 55 and 70 oC ...... 60

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LIST OF TABLES Table 1. Benefits of Ethyl Lactate ...... 3 2. Information about the Chemicals Used: Name, Chemical Structure, CAS number, % Purity and Molecular Weight ...... 25 3. Polarimeter Operating Conditions ...... 27 4. GC-MS Operating Conditions ...... 28 5. Optical Activity Results at T= 20 oC ...... 29 6. Refractive Index Results on Ethyl Lactate Enantiomeric Mixtures ...... 32 7. Weight Fraction of Ethyl Lactate Enantiomeric Mixture vs. Observed Melting o Temperatures, Tm, ( C) using the Modulated-DSC Technique ...... 34 8. Important Thermodynamic-Parameters that were Determined from the Binary Phase Diagram of Ethyl Lactate Enantiomers using the Modulated-DSC Technique ...... 34

9. Reichardt’s ET(30) Polarity Scale for (-)-Ethyl L-Lactate, (+)-Ethyl D-Lactate and Racemic Mixture at T=10-70 oC ...... 36 10. Hydrogen-Bond Acceptance (Basicity) for (-)-Ethyl L-Lactate, (+)-Ethyl D-Lactate and Racemic Mixture at T=10-70 oC ...... 38 11. Hydrogen-Bond Donation (Acidity) for (-)-Ethyl L-Lactate, (+)-Ethyl D-Lactate and Racemic Mixture at T=10-70 oC ...... 39 12. Polarizability for (-)-Ethyl L-Lactate, (+)-Ethyl D-Lactate and Racemic Mixture at T=10-70 oC ...... 41 13. Reichardt’s and Kamlet-Taft Parameter Results for (-)-Ethyl L-Lactate ...... 60 14. Reichardt’s and Kamlet-Taft Parameter Results for (+)-Ethyl D-Lactate ...... 60 15. Reichardt’s and Kamlet-Taft Parameter Results for Racemic Ethyl Lactate Mixture ...... 61

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INTRODUCTION

Molecules with stereocenters are called chiral compounds and they will have enantiomers (non-superimposable mirror images). An interesting phenomenon occurs when a chiral molecule is subjected to a plane-polarized light. A plane of polarized light rotates when it strikes the chiral molecule. If that rotation occurs in a clockwise direction then that rotation is classified as (+). A counterclockwise rotation is (-). A racemic mixture (50:50 mixture of both enantiomers) gets (±) in the beginning of the name to indicate that both enantiomers exist simultaneously. The racemic mixture will not rotate polarized light. Another classification that is most commonly used to classify amino acids and sugar is the (D)-/(L)- system. Where (D)- is the dextrorotary or right handed enantiomer and (L)- is the levorotary or left handed enantiomer.

A mixture containing 50:50 amounts of both (D)-/L(-) enantiomers is called a racemate.

Organic esters have various applications in the following fields of chemical industry: perfumes, flavors, pharmaceuticals, plasticizers, solvents and intermediates [1]. Ethyl lactate, also known as Lactic acid ethyl ester, is a monobasic ester (structure shown in figure 2). The

IUPAC name is Ethyl (S)-2-hydroxypropanoate, with a molecular formula of C5H10O3. In small quantities it is found in a variety of foods, such as wine, chicken and some fruits. It is a clear to slightly yellow liquid at room temperature. A reversible reaction between ethanol and lactic acid produces ethyl lactate at an industrial level. The L-form of the ethyl lactate is less toxic, more commonly manufactured, and is cheaper than the D-form [2].

Figure 1. The Molecular Structure of Ethyl Lactate [1]

Ethyl Lactate is a polar, protic solvent, has high boiling point, and is considered a green organic solvent. It is produced industrially from corn, a renewable feedstock. In addition to all the environmental and toxicology favorable benefits of ethyl lactate listed in table 1, it also possesses excellent solvent properties [3]. The vicinity of hydroxyl and carbonyl groups contained in ethyl lactate structure give rise to remarkable intramolecular hydrogen bonding.

The hydroxyl group in ethyl lactate exhibits a hydrogen bond donor site, whereas the ester functional group in the molecule exhibits the hydrogen bond acceptor site. These remarkable properties of ethyl lactate make it an excellent solvent of choice in flavor additive, degreasing, pharmaceutical drug synthesis, manufacturing of cosmetics products, dissolving resins mixtures, and acetic acid cellulose solutions [4].

Table 1: Benefits of Ethyl Lactate

Major Advantages of Ethyl Lactate

Non-Carcinogenic High Boiling Point

High Boiling Point 100 % Biodegradable

Non-Corrosive Easy/Inexpensive to recycle

Flavor additive Stable in Solvent form approved by FDA unless exposed to water

Green Solvent Does not deplete ozone

Ethyl lactate, as evident from figure 2, is a chiral molecule because of its two stable enantiomeric forms: (-)-Ethyl L-Lactate (left side of figure 2) and (+)-Ethyl D-Lactate (right side of figure 2).

Figure 2: Two Enantiomeric forms of Ethyl Lactate [5] [6]

Despite the potential applications for ethyl lactate and many other chemicals from the lactate family, no in-depth studies have been conducted to measure physical of ethyl lactate such as: density, viscosity and refractive index [7]. Melting point, Tm, of ethyl lactate is documented incorrectly in various MSDS literatures, such as Sigma-Aldrich and Acros

Organics. Consulting the literature creates confusion and shows inconsistent results both in terms of melting point and phase diagram of (-)-Ethyl L-Lactate. Based on the literature findings, it appears that the documented melting point of pure (-)-Ethyl L-lactate; Tm= -26 oC does not correspond to pure L-form of ethyl lactate. Further, there is very limited to none information on basic physical and chemical properties of (+)-Ethyl D-Lactate in literature such as melting point, refractive index, specific gravity, acidity etc.

Objective

Due to the discrepancy in the literature data on physical properties (melting point, density, optical rotation, refractive index) of enantiomeric mixtures of (-)-Ethyl L-Lactate and (+)-

Ethyl D-Lactate investigate the following properties of ethyl lactate enantiomeric mixtures:

Optical rotation, [α] , Density (ρ), refractive index (nD) at 293.15 K, binary phase diagram of the ethyl lactate enantiomers using differential scanning calorimetry (DSC) to characterize the melting point, Tm, of (-)-Ethyl-L- Lactate, (+)-Ethyl-D-Lactate, racemic mixture, and eutectic point

(the lowest melting point of ethyl lactate enantiomeric mixture, and Solvents Properties

(Solvatochromism) at T= (10-70) °C.

Gas Chromatography-Mass Spectrometery (GC-MS) will be used to analyze the purity of

L and D enantiomers of ethyl lactate.

There is no clear data available in literature that lists solvatochromic properties of L- form, D-form and racemic ethyl lactate mixtures. Only one study used Reichardt’s betaine dye to document the solvent polarity scale of (-)-Ethyl L-Lactate [8]. The current study was conducted in part to determine the solvatochromic characterization of ethyl lactate enantiomers and the racemic mixture. The aim is to get the comprehensive and up-to-date collections of solvatochromic parameters.

Optical Rotation

Enantiomeric purity of an optically active chiral compound can be monitored through its optical rotation. The optical rotation of a chiral compound is reported in terms of a specific rotation, [α], as shown below in equation 1.

 Specific rotation = [α] = (Equation 1) c x l

Where [α] is the specific rotation, α is the observed rotation, c is the concentration

(measured in g/mL), and l is the length of the light path through which it passes through the solution (measured in decimeter).

For neat liquids, equation 2 is used to calculate the specific rotation:

 Specific rotation = [α] = (Equation 2) l x d

Where [α] is the specific rotation, α is the observed rotation, l is the length of the light path through which it passes through the solution, and d is the density of the liquid measured in g/mL. An apparatus such as pycnometer can measure the density of a liquid with the high degree of accuracy.

The specific rotation of a molecule is measured in a lab through the device called a polarimeter (as shown in figure 4). The light source used in polarimeter is a sodium lamp that emits light at a specific wavelength, λ = 589 nm, which is also called the sodium D-line.

Instrumental polarimeter measurements are typically reported as α/ℓ.

Figure 3. Schematic Diagram of a Polarimeter [9]

The specific rotation of a compound is also influenced by temperature and wavelength of the light used. However, these two variables cannot be included in the specific rotation equations (Equations 1 & 2) because there is a non-linear correlation between them. Therefore, these two variables: Temperature, T and wavelength, λ, are formatted the following way to

T represent the specific rotation: []D where T is the temperature of the cell (in degree Celsius).

Most literature report T as 20 oC. D is the line of sodium that emits light at λ = 589 nm.

A single enantiomer in a solution categorizes it as an optically pure solution. This implies that another enantiomer is entirely absent. However, when a solution contain equal amounts of both enantiomers (racemic mixture), in a 50:50 ratio, it becomes an optically inactive solution.

When such solution is subject to the polarimeter analysis, the net rotation becomes zero.

A solution containing both enantiomers in an unequal amount will be optically active.

Such solution is optically impure (containing both enantiomers). In that case, optical purity of that solution is defined in terms of percent enantiomeric excess via following equations:

|observed []| Optical purity = x 100 % [9] (Equation 3) |[]of pure enantiomer |

d  l Optical purity = % ee = x100% (Equation 4) d  l

By using equation 4, one can determine the relative proportions of each enantiomer in a mixture that is not optically pure (contain both L and D enantiomers).

Density (ρ):

Literature density values of enantiomers of Ethyl lactate are very scarce. MSDS doesn’t even report the density of (+)-Ethyl-D-Lactate. Therefore, experimental density data has to be obtained for characterization of enantiomeric mixtures of Ethyl Lactate. A device called a pycnometer can accurately measure the density of a fluid by using an analytical balance. A pycnometer is made up of glass containing a close-fitting glass stopper that contains a capillary tube through it that lets the air bubbles escape from the apparatus. Using water as an appropriate reference working fluid, pycnometer can measure density, ρ, of a substance very accurately. The following equation is used to find the exact volume of the pycnometer:

m H O  = 2 (Equation 4) H 2O V

o Where ρ H2O = Density of water at 20 C

mH2O = mass of water

V= volume of pycnometer

Once the pycnometer volume is calibrated, it can now measure the density of the liquid using the following equation:

m   L x  (Equation 5) L m H2O H2O

This study will use a 2-mL pycnometer to measure the densities of (-)-Ethyl-L- lactate,

(+)-Ethyl-D-Lactate, its racemic mixture along with various enantiomeric mixtures of Ethyl

Lactate at T=20 oC.

Refractive Index (nD):

Similar to density, nD is a bulk-thermophysical solvent parameter that is useful in predicting solvent characteristics, such as polarity, hydration and percent purity. This study will determine the refractive index of various binary mixtures of ethyl lactate enantiomers. Once the experimental results of nD are obtained they will be compared against the literature values to see if there is an agreement.

Differential Scanning Calorimeter (DSC):

Differential Scanning Calorimetry (DSC) is a calorimetric technique which measures the difference in a heat flow rate between the sample and inert reference as a function of temperature and time. DSC is the most commonly used thermal analysis quantitative technique through which following information can be obtained: purity of a sample, melting point (Tm), heat of fusion, % Crystallinity, crystallization time and temperature, glass transition (Tg), specific heat

(Cp), degree of cure, and thermal stability.

DSC instrument has a sample and a reference that are placed in the instrumental- holder. The instrument measures the difference in the heat flow (mW =mJ/sec) between the sample and the inert reference as a function of time and temperature. The output from DSC, thermogram, plots the following information: heat flow rate (W/g) vs. Temperature (oC).

DSC instrumentation technique is categorized into three types: Power Compensated DSC,

Heat-Flux DSC, and Modulated DSC technique [10].

In a power-compensated DSC, the temperature of the sample and reference are kept equal to each other. Both temperatures increase or decrease linearly. The power needed to maintain the sample temperature equal to the reference temperature is measured in this analysis. Power-Compensated DSC has lower sensitivity than heat-flux and modulated DSC.

However, power-compensated DSC has higher resolution than the other two techniques. This makes power-compensated DSC suitable for kinetics studies in which fast equilibrations to new temperature settings are needed.

Heat-flux DSC measures the difference in the heat flow between the sample and the reference at a constant rate. Heat flows into sample as well as the reference via electrically heated thermoelectric disk.

In a Modulated DSC, heating arrangement as well as cell arrangements are identical to the heat-flux DSC technique. Modulated DSC uses a sinusoidal program to produce a microheating and cooling cycle as the overall temperature is continually increased or decreased. Modulated DSC uses a fourier transform method to split the signal into two parts: reversible heat flow signal and non-reversible heat flow signal. Reversible heat flow contains thermodynamic information in modulated DSC, such as Tg and Tm, whereas the nonreversible heat flow contains kinetic information, such as curing, decomposition, crystallization and sometimes melting.

The following important thermal events are seen in a typical DSC thermogram, and they are useful in characterizing the important thermal properties of a substance:

- Exothermic Transition: An upward pointing peak (maxima) indicates an exothermic

event. Examples of exothermic events detected by DSC include oxidization,

decomposition, crystallization etc.

- Endothermic Transition: A downward pointing peak (minima) indicates an endothermic

event. Example of endothermic events in DSC are melting point, evaporation etc.

- Glass Transition, Tg: A transition from an amorphous state to a glassy, brittle one. The

following factors affect the glass transition: heating rate, molecular weight, chemical

structure and physical aging of a molecule. In a modulated DSC, Tg is difficult to detect

because it only happens in a reversible heat flow of the signal. Using a larger sample

weight and slow scan rate also makes the glass transition difficult to detect.

- Melting Point, Tm: An endothermic transition from crystalline phase to amorphous state is

represented by Tm. Tm is affected by molecular weight, chemical structure, and additives.

The melting endotherms of a pure substance are very sharp i.e. they show narrow

temperature range interval [11]. For impure substances, the melting endotherms are

broader and indicate impurity contents [11].

- Enthalpy of Melting, ΔHm: The heat required for melting (also known as heat of fusion).

This is calculated by integrating the area of DSC peak on a time basis.

- Crystallization temperature, Tc: An exothermic transition results in cooling from liquid to

crystalline solid. ΔHm gives important information about the % Crystallization through the

following formula:

H (exp) % Crystallization= m (Equation 6) H o

In equation 6, ΔHm = experimental enthalpy of melting or fusion and ΔHo= literature value

for the enthalpy of fusion of a purely crystalline sample.

- Enthalpy of Crystallization, ΔHc: The amount of heat released when crystallization

happens. Similar to ΔHm, it is calculated by integrating the area of DSC peak on a time

basis.

- Binary Phase diagram of enantiomers (as shown in figure 4 below) to determine the

eutectic point (lowest Tm corresponding to % composition of the binary mixture).

Figure 4. Binary Phase Diagram of Enantiomers showing the Eutectic Point [12]

A sample thermogram of DSC as shown in figure 5, gives a visual representation of important thermal events (I-VII).

Figure 5. DSC Thermogram showing various Thermal Events [13] The application of DSC in constructing the binary phase diagrams of enantiomeric mixtures is a widely used procedure in characterization of racemic drugs [14]. In this study, modulated-DSC technique will be used to investigate the following: binary phase diagram of ethyl lactate enantiomers, determine the lowest Tm corresponding to eutectic weight % of ethyl lactate enantiomers, investigate the existence of metastable phase equilibria from the phase diagram, and Tm of the racemate.

From a theoretical stand point, based on the binary phase diagram of enantiomeric mixtures and DSC output (thermogram), melting temperature (Tm) can be related to mole fraction (x) and enthalpy of fusion (ΔHm) by using the following classical thermodynamics, Van’t

Hoff equation:

H m  1 1  ln x     [15] (Equation 7) R Tm T 

For the racemate, its melting point, enthalpy of fusion and mole fraction can be related by

Prigogine-Defay equation:

2H   m,rac  1 1  ln 4x(1 x)     [15] (Equation 8) R Tm,rac T 

Solvatochromism

Most chemical reactions are carried out in solution. Success of that chemical reaction is dependent on the selection of appropriate solvent. Solvents strongly affects the position of chemical equilibria, reaction rates, the position and intensity of spectral absorptions such as:

UV/Vis, IR, ESR, NMR [16]. For instance, the solvation of 2-chloro-2-methylpropane via SN1 mechanism is 1011 times faster in water (polar solvent) than in benzene (non-polar solvent) as a result of solvent effect [17]. Understanding the solvent polarity and hydrogen bonding abilities helps us understand the solvent effect.

Solute-solvent interaction leads to the origin of solvent polarity. Certain thermo-physical solvent properties such as refractive index, density, boiling point, viscosity, or relative permittivity are helpful in understanding and predicting solvent polarity. However, those properties do not help in understanding the overall solute-solvent interaction such as: van der

Waals interaction, hydrogen-bond donation and acceptance abilities of a solvent that occurs at a molecular, microscopic level. Solvent polarity is the overall solvation ability of a solvent, which depends on solvent-solvent and solute-solvent interaction [18].

Solvent polarity from a theoretical stand point is the overall solvation capability of solvents which depends on the intermolecular forces between the solute and the surrounding solvent molecules [18]. Those intermolecular force interactions cause a change in the electronic spectrum (UV- Vis spectra). Polarity of solvents can hardly be defined by using a single parameter [19]. Thus, one needs to analyze solvent polarity in terms of acidity, basicity and dipole effects.

In literature, the most commonly used methods to investigate solvent polarity are the ones suggested by Reichardt and Kamlet-Taft. They suggested that polarity of a solvent can be best understood by empirically measuring dipolarity/polarizability (π*), hydrogen-bond donating

16 ability (α), and hydrogen-bond accepting ability (β) of a solvent by using various types of probe- dyes. Some of the more common dyes used are Reichardt’s Betaine dye, N,N-diethyl-4- nitroaniline, and 4-nitroaniline (p-nitroaniline). The molecular structure of these solvatochromic probe dyes are shown in figure 6.

Reichardt’s Betaine Dye p-Nitroaniline (pNA) N,N-diethyl-4-nitroaniline (DENA)

Figure 6. Structures of Solvatochromic Probes used in this study to Investigate Solvent Polarity and Hydrogen-Bonding Characteristics of Solvents [20]

All of those variables are important when predicting polarity: Polarizability/depolarization,

π*, gives direct information about the Van der Waals Interaction between solute-solvent. π*, indicates the ability of a solvent to stabilize the negative charge between solute-solvent [21].

Hydrogen bond donating-acidity, α, of the solvent provides a relative measure of the proton donating ability of the solvent. Hydrogen bond accepting-basicity, β, of the solvent provides a relative measure of the proton accepting ability of the solvent. Molar transition energy of maximum absorbance, ET(30) formally known as solvent polarity scale, provides information on both the polarizability of the solvent as well as its hydrogen bond donating ability.

Reichardt’s Betaine dye: 2,6-Diphenyl-4-(2,4,6-triphenyl-1-pyridinio) phenolate is a zwitterionic solvatochromic dye with a permanent dipole as shown in figure 6. Reichardt’s

17 betaine dye is strongly sensitive to the polarity and hydrogen bonding donor ability (HBD) of the solvent. When HBD ability of the solvent along with the polarity increases, the dye shows large spectral band position change. This feature makes Reichardt’s dye the most suitable dye in

N determination of empirical solvent parameter formally called ET (30) and its normalized form ET through electronic spectroscopy (i.e. UV/Vis Spectroscopy). The position of its longest- wavelength intramolecular charge-transfer absorption band depends on solvent polarity, solution temperature, external pressure, and the nature and concentration of added salts [16].

The absorption type of that longest-wavelength intramolecular charge-transfer absorption band is π → π*, and is detected by UV-Vis spectroscopy.

Zwitterionic Reichardt’s Betaine dye, is a sensitive solvent polarity indicator dye because of its ability to measure the solute-solvent interaction. This interaction is due to the following structural features of this dye:

- Highly dipolar electronic ground state, µG= 15 Debye

- Aromaticity: localized, π-system consist of 44 π electrons

- Phenolate oxygen that shows strong electron pair donor or strong hydrogen bond

accepting site. This site is suitable for interacting with hydrogen bond donor (HBD)

solvents in making hydrogen bond especially with with protic solvent [16]

- The dye’s Zwitterionic behavior (as seen from its structure in figure 6). If protonated, it

will no longer be functional

Figure 7. Molecular Structure of the Zwitterionic Reichardt’s Betaine Dye showing its Inherent Properties [16]

When the electronegative, HBA site of the zwitterionic Reichardt’s Betaine dye (as shown in figure 7) interacts with the HBD site of a solvent, solvatochromic shift is induced due to the intramolecular transfer of electrons. UV-Vis Absorption spectra registers this electronic transition by giving the position of the maximum absorbance [21]. The molar transition energy in this process is called ET(30) and is defined by equation 18. The positive charge in Reichardt’s dye is delocalized and shielded by 2,4,6-phenyl groups that makes the pyridine ring a weak electron pair acceptor or hydrogen bond donor site [16]. Therefore, hydrogen-bond acceptor (β)

(basicity) of solvents cannot be measured using Reichardt’s dye.

Reichardt’s Betaine Dye must remain deprotonated in a solution to function effectively as solvatochromic indicator [21]. In acidic solvent, the phenolate oxygen atom of the Reichardt’s dye gets protonated. This blocks the intramolecular charge transfer process, which is the crucial

19 step in initiating solvatochromism. As a result, the charge transfer band will not be observed in the UV-Vis Absorption spectra [22]. To overcome this problem, another zwitterionic, but slightly basic, dichloro-substituted Reichardt’s Betaine dye, as shown in figure 8, can be used. The pKa of Reichardt’s Betaine dye in literature is 8.6 [23]. The pKa of the dichloro-substituted

Reichardt’s dye is 4.8 [21].

Figure 8. Molecular Structure of Dichloro-Substituted Reichardt’s Betaine Dye [23]

4-Nitroaniline: Another solvatochromic probe dye that helps investigate the HBA/HBD ability of a solvent is 4-nitroaniline. Based on its molecular structure the amino functional group in 4-nitroaniline has hydrogen-bond donating ability (HBD) while the nitro group has hydrogen- bond accepting ability (HBA). When it is solvated with hydrogen-bond accepting solvent, hydrogen-bond is formed that causes the charge transfer and stimulates the solvatochromism.

The solvatochromism intramolecular charge transfer band in 4-nitroaniline occurs at around

320-388 nm, when the dye is solvated with solvents of varying polarity [24]. When 4-nitroaniline is solvated with binary solvents, the intramolecular charge transfer band is unaffected by the

20 isobestic point [24].This indicates that the two resonance forms of 4-nitroaniline, as shown below in figure 9, does not contribute in band shifting in electronic spectroscopy.

Figure 9. Resonance Forms of 4-Nitroaniline [24]

Based on the structure of 4-nitroaniline, it is clear that (i) any hydrogen-bond accepting

(HBA) solvent can form a strong hydrogen-bond with the amino hydrogens and (ii) any hydrogen-bond donating (HBD) solvent can form a strong hydrogen bond with the nitro oxygens. These bonding interactions are expressed in figure 10.

Figure 10. HBD and HBA Sites in 4-Nitroaniline [24]

N,N-diethyl-4-nitroaniline: Based on the structure of N,N-diethyl-4-nitroaniline, it is clear that any hydrogen-bond donating (HBD) solvent can interact and form a strong hydrogen-bond with the nitro functional group, but there are no longer any protons to interact with hydrogen-

21 bond accepting (HBA) solvent. Therefore, N,N-diethyl-4-nitroaniline only has hydrogen-bond acceptance (HBA) ability as illustrated in figure 12.

Figure 11. HBA Sites in N,N-Diethyl-4-Nitroaniline [25]

In comparison, both dyes: 4-nitroaniline & N,N-diethyl-4-nitroaniline exhibit HBA ability through the NO2 functional group. However, only 4-nitroaniline has the HBD ability through the amine, NH2, functional group as shown in figure 11.

All of the three solvent probe dyes: Reichardt’s Betaine dye, 4-Nitroaniline, and N,N- diethyl-4-nitroaniline are thermochromics, which means that they can be used to predict solvent properties at elevated temperatures.

Equations Used to Measure Solvatochromic Parameters:

The Polarizability/Dipolarity (*) parameter is determined using N,N-diethyl-4-nitroaniline

(DENA) through the following equation suggested in the literature.

10000 27.52   *= DENA (Equation 9) [26] 3.182

λDENA = Maximum wavelength (nm) detected from the UV-Vis Spectroscopy induced by Solvatochromism.

The ET(30) scale that Reichardt’s suggested can indicate solvent depolarization and hydrogen- bond donation ability by using equation 10:

α= -1.47 – 0.532π* + 0.0508 ET(30) (Equation 10) [22]

Where ET(30) = h c υmax NA (Equation 11) [18]

ET(30) = Intramolecular charge-transfer energy when Reichardt’s Betain dye is mixed with a solvent. It is called Polarity Scale and is measured in kcal/mol h= Plancks Constant (6.626 x 10-34 J.s) c= speed of light (3 x 108 cm/s)

23 NA = Avagadro’s number (6.023 x 10 photons/mol)

-1 υmax= 1/λmax (cm ) Note: π* in equation 10 is from equation 9. Following equation is applied when calculating the hydrogen bond acceptance, β, of solvent.

1 1 (1.035  2.64 ) β=  DENA  PNA [27] (Equation 12) 2.80

Where λ DENA and λpNA are the wavelength (nm) corresponding to the maximum absorbance values in N,N-diethyl-4-nitroaniline and p-nitroaniline dyes respectively.

Gas Chromatography-Mass Spectrometry (GC-MS):

Combination of Gas-Chromatography (GC) and Mass Spectrometery (MS) is an effective combination in chemical analysis such as finding % purity, drug testing and evaluating the extent of separation in mixtures [28]. The GC separates the components based on their relative attraction towards the solid phase column and is expressed in terms of the retention time. The MS part provides various possibilities in chemical identity and is expressed in terms of mass/charge ratio. This study will use GC-MS to analyze the purity of (-)-Ethyl L-Lactate, (+)-

Ethyl D-Lactate and racemic mixture.

EXPERIMENTAL SECTION

The chemicals used in this study and their purities are provided in Table 2. Table 2. Information on Chemicals Used: Name, Chemical Structure, CAS number, % Purity and Molecular Weight

UV-Vis Spectroscopy: UV-Vis absorption spectra were recorded in the range 200–800 nm at the following temperatures (in degree Celsius): 10, 25, 40, 55 and 70 with a CPS-240A

UV-2550 spectrophotometer. Slow scan speed was selected and sampling interval (nm) was 1.

Each dye was prepared in concentration of 0.001 (± 0.0005) g/mL. The volume of solvent was

1000 µL. Quarts cuvette with a path cell length of 10.00 mm was used to place the solution in the cell holder. Perkin Elmer, Peltier Temperature Programmer, PTP 1 was used to control the cell temperature to ± 0.1 oC.

Differential Scanning Calorimetry (DSC): The DSC data were collected with DSC Q 2000

V24.9 Build 121 calorimeter. Modulated DSC technique was used for data collection. Samples

25 were equilibrated at 10.0 oC. Modulated rate was 0.053 oC for every 40 s. Isotherm conditions for 5.00 min were selected. Ramping was done from 0.500 oC/min to -70 oC and 0.500 oC/min to

10 oC. Flange temperature was allowed to reach at -80 oC before the runs were initiated.

Ethyl Lactate samples with enantiomeric composition ranging from 0:100 [(L)/(D)] to

100:0 [(L)/(D)] were prepared, including 50:50. Samples were prepared in T-zero pans with hermetic lids. The sample mass was 11 (±1.5) mg. The reference pan was Tzero pan with hermetic lid that had a mass of 52.48 mg.

Density: Density determination was completed by using a 2-mL pycnometer at 20oC.

HPLC graded water was used to calibrate the pycnometer at 20oC. All density measurements were recorded in g/mL units. The temperature dependent density of water, ρ(T=20 oC) =

0.99820 g/mL was used as a correction factor in calibration.

Density Measurements were taken 3 times per ethyl lactate sample (n=3), and summarized in table 5. Average density, x mean was recorded per sample. Standard deviation, s, of each sample was calculated by using the following formula:

 (x  x ) s  i mean (Equation 13). n 1

Refractive Index (nD) Determination: RHB-62-ATC Brix, hand-held refractometer was used to get the Brix % values at 20 oC. Brix % values were converted to refractive index values.

Optical Measurements: A Rudolph Research Analytical Autopol IV polarimeter housed at the US Naval Academy, Annapolis, MD, was used to measure the refractive index. The following were the operating conditions used for the polarimeter.

Table 3: Polarimeter Operating Conditions Operating Cell Temperature 20 oC Wavelength (nm) for D-Line of 589 nm Sodium Response Time 2-sec Quartz Control Plate Cell Description Calibration Standard Rotation at 20◦C and 589 nm, 11.491 degrees accepted value Rotation at 20 °C and 589 nm, 11.491 degrees measured value Part Number of Polarimeter 14-4.0-100-1.5

Gas Chromatography-Mass Spectrometery (GC-MS): Agilent technology GC-MS instrumentation was used with the following parameters to run the GC-MS on (-)-Ethyl L-

Lactate, (+)-Ethyl D-Lactate, and racemic ethyl lactate mixture.

Table 4: GC-MS Instrumental Operating Conditions Oven Temperature 60 °C Hold time 3.00 min Run Time 3.00 min Column Pressure 1.48 Flow Rate 1 mL/min Average Velocity 51 cm/sec Mode Constant Flow Gas Carrier (mobile Helium (He) phase) Heater Temperature 250 °C Pressure 0.44 psi Split Ratio 100/1 Split Flow 99.4 mL/min Minimum Peak Width 1Hz, 2 min Inlet Pressure, psi 0.44 psi Total Inlet Flow 103 mL/min Equilibration Time 0.50 min Maximum 350 °C Temperature Injection Volume 1 µL Syringe Size 10 µL

RESULTS AND DISCUSSION:

Optical Rotation [α] and Density (ρ) Results:

The densities and optical rotation measurements for enantiomeric mixtures of ethyl lactate are shown in Table 5. Table 5: Optical Activity Results at T= 20 oC

Composition of Ethyl (α/Ɩ)Blank in (α/Ɩ)Sample in αObs /Ɩ (deg/cm) Mean density, [α] = α/Ɩd Lactate Enantiomers degrees degrees d (g/mL) (degrees)

100% L, 0% D-Ethyl Lactate -0.002 -10.196 -10.194 1.028 -9.916

80% L, 20% D-Ethyl Lactate 0.005 -6.161 -6.166 1.025 -6.016

60% L, 40% D-Ethyl Lactate 0.003 -1.708 -1.711 1.026 -1.668

50% L, 50% D-Ethyl Lactate 0.005 0.627 0.622 1.026 0.606

40% L, 60% D-Ethyl Lactate 0.005 2.615 2.61 1.021 2.556

20% L, 80% D-Ethyl Lactate 0.013 7.108 7.095 1.025 6.922

100 % D, 0% L-Ethyl Lactate -0.014 11.4 11.414 1.022 11.168

In literature, (-)-Ethyl L-Lactate, at T= 20 oC, has documented specific optical rotation, [α] value of -10.5 degrees (neat) [5]. Experimental optical activity value for (-)-Ethyl L-lactate, whose purity was ≥ 99.0% has an experimental [α] value of -9.915 degrees that yields 5.6 % error. The [α] value for the racemic mixture was 0.606. The specific optical rotation for (+)-Ethyl

D-Lactate was 11.163 degrees. The most intuitive possibility is an excess amount of (+)-Ethyl

D-Lactate in the pure L-sample. However, it was noted after the measurement that the vials used had rubber cap liners, which was slowly dissolving in the ethyl lactate solutions.

Unfortunately, there was not enough time to gain access again at the U.S. Naval Academy to repeat the results in Teflon lined vials, which were used for all other measurements.

Furthermore, the L-form of ethyl lactate used in this study was at least 98% pure, while the D-form was only 95% pure. In light of this, the higher optical activity for the D-form suggests that the impurity might be a chiral compound, with an optical activity that is greater than +11. D- form could be enantiomerically impure by either another solvent or a combination of enantiomeric solvents. Further determination can be made to affirm this by using chiral column

HPLC or GC-MS analysis. To determine the relative purities of the two forms and elucidate the identity of the impurity, GC-MS analysis was performed.

Gas Chromatography-Mass Spectrometry (GC-MS) Results:

GC chromatograms and MS spectra of each significant chromatograph peak are provided in Appendix A. GC Chromatogram of (-)-Ethyl L-Lactate shows only 1 predominant peak in the chromatogram. The GC chromatogram of (+)-Ethyl D-Lactate contains three significant peaks. For the racemic mixture, the GC chromatogram also contains three peaks.

NIST library search results for the MS spectrum of the first peak matches the spectrum for ethyl lactate. The MS spectrum in the small second peak for (-)-Ethyl L-Lactate show the presence of hydroxybutyric acid with a retention time of 6.0 min. The second peak in the (+)-Ethyl D-Lactate and in the racemic ethyl lactate solution has a retention time of 6.5 min and may contain some

2-butanol. This suggests the presence of 2-butyl lactate ester or ethyl 2-hydroxybuyrate ester as impurities. Since the lactate esters of higher molecular weight alcohols have higher optical rotations,[29] this result is consistent with the observed optical rotation values obtained. The third peak indicates that there is also a trace impurity of a higher molecular weight compound.

The NIST library search result suggests a glycol oligomer, such as hexaethylene glycol.

Refractive Index (nD) Results:

Table 6: Refractive Index Data of Ethyl Lactate Enantiomeric Mixtures Ethyl Lactate Mixture Composition Brinx % at T=20 °C Index of Refraction, nD 100 % L, 0% D-form 49.1 1.4181 80% L, 20% D-form 49.2 1.4183 60% L, 40% D-form 49.2 1.4183 50% L, 50% D-form 49.3 1.4185 40% L, 60% D-form 48.95 1.4178 20% L, 80% D-form 49 1.4179 100% D, 0% L-form 49.08 1.4181

Experimental refractive index (nD) value of (-)-Ethyl L-Lactate from table 6 are in close agreement with the literature documented nD = 1.4130 value of (-)-Ethyl L-Lactate [5]. All refractive index values are within experimental error, suggesting that the refractive index is not affected by the chirality or enantiomeric excess of the solvent.

Differential Scanning Calorimetry (DSC) Results:

Figure 12: Binary Phase Diagram of Ethyl lactate Enantiomers The temperatures of the phase transition exotherms observed upon heating were measured using modulated DSC and are reported in Table 7. The heating curves showing these transitions is provided in Appendix B. Using these temperatures, a phase diagram was constructed for enantiomeric mixtures of ethyl lactate, as shown in Figure 12. Slow heating and cooling rates obtained thorough the modulated DSC technique provides an accurate way to construct phase diagram [11].

Table 7: Weight Fraction of Ethyl Lactate Enantiomeric Mixture vs. Observed Melting o Temperatures, Tm, ( C) using the modulated-DSC Technique

Wt % (-)-Ethyl-L-Lactate Eutectic Temp I ( °C ) Eutectic Temp II ( °C ) Tm of Mixture ( °C) 0 -4.23 0.1 -39.57 -27.01 -10.62 0.2 -40.57 -25.33 -15.63 0.3 -42.13 -24.49 -20.55 0.4 -41.8 -24.33 -22.9 0.5 -25.64 0.6 -26.95 0.7 -41.35 -25.72 -18.06 0.8 -45.94 -28.77 -14.83 0.9 -45.71 -30.92 -11.04 1 -6.22

Table 8: Important Thermodynamic-Parameters determined from Binary Phase Diagram of Ethyl Lactate Enantiomers using the Modulated DSC Technique o o Eutectic Temperature (Te) in C -26.95 C Eutectic Composition, Wt. % 60 % (-)-Ethyl L-Lactate Enthalpy of Fusion at Eutectic Point 2.194 J/g Melting Point, Tm of Racemate -25.64 oC Enthalpy of Fusion of Racemate 83.46 J/g Melting Point, Tm of (-)-Ethyl L-Lactate -6.22 oC Enthalpy of Fusion of (-)-Ethyl L-Lactate 40.12 J/g Melting Point, Tm of (+)-Ethyl D-Lactate -4.23 oC Enthalpy of Fusion of (+)-Ethyl D-Lactate 159.4 J/g From a theoretical stand point, an extension of enantiomer liquidus lines intersecting at racemic composition depicts the position of a metastable equilibrium [30]. Phase diagram of binary system of ethyl lactate enantiomers reveals that all of the melting point curves seem to

o converge at a eutectic point in 60:40 [(L-form)/(D-form] at Te = -26.95 C.

o The melting point of racemate was Tm= -25.64 C. This Tm value of racemate matches

o with the literature documented Tm (-26 C) of (-)-Ethyl L-lactate on manufacturer’s MSDS. The

o actual Tm of (-)-ethyl l-lactate, based on this study, was at -6.22 C. Comparing this value with the literature Tm of (-)-Ethyl L-lactate gives a percent error of 76 %. Therefore, there is a significant evidence to believe that the literature documented melting point of (-)-Ethyl L-lactate

34 was a racemate of ethyl lactate solvent. There is also the presence of a solid – solid transition at around -40 °C, where the crystal structure of the racemate changes.

Solvatochromism Results: The UV-Vis spectra of the three solvatochromic dyes in (-)-Ethyl L-Lactate, (+)-Ethyl D-

Lactate and Racemic Mixture can be found in Appendix C. From the peak wavelengths, the solvatochromic parameters for these solvents were calculated. The variation of the ET(30) polarity scale with temperature for the ethyl lactate solvents was calculated from equation 11 and is shown in Table 9 and Figure 13.

Table 9. Reichardt’s ET(30) Polarity Scale for (-)-Ethyl L-Lactate, (+)-Ethyl D-Lactate and Racemic Mixture at T=10-70 oC

T (K) ET(30) for (-)-Ethyl L-Lactate ET(30) for (+)-Ethyl D-Lactate in ET(30) for Racemic Mixture kcal/mol kcal/mol in kcal/mol 283 57.41 58.35 58.11 298 57.30 57.99 58.23 313 56.95 58.35 58.23 328 56.73 58.11 58.11 343 56.73 57.99 58.47

58.6

58.4

58.2

(30) (30) 57.8

T T E 57.6

57.4

57.2 (-)Ethyl-L-Lactate 57 (+)Ethyl-D-Lactate 56.8 Ethyl-D,L-Lactate 56.6 250 260 270 280 290 300 310 320 330 340 350 T (K)

Figure 13: Plot Showing Temperature Dependence of ET(30) Polarity Scale in (-)-Ethyl L- Lactate, (+)-Ethyl D-Lactate and Racemic Ethyl-D,L-Lactate Mixture

Figure 13 shows that the ET(30) values, in kilocalories per mol, when Reichardt’s dye was used to measure ET(30) scale for all three solvents. The trend observed in figure 13 shows that that ET(30) parameter decreases for the L-form of ethyl lactate but is relatively constant for the D-form and racemic mixture. This is opposite to the literature findings [22].

Reichardt’s dye could be protonated in ethyl lactate solutions, which makes the dye incapable of doing intramolecular charge transfer in (-)-Ethyl L- Lactate, (+)-Ethyl D-Lactate and racemic ethyl lactate solvents. Furthermore, UV-Vis spectra of (-)-Ethyl L-lactate, (+)-Ethyl D-

Lactate and racemic mixture do not show any clear electronic transition maxima. MSDS reports that for (-)-Ethyl L-Lactate is slightly acidic (pH=4) [5]. This clearly indicates that Reichardt’s dye was not a suitable dye in studying polarities due to the acidity in the solution.

The effectiveness of Reichardt’s dye can be re-evaluated for ethyl lactate enantiomeric solvents by recrystallizing the dye prior to solvatochromic measurements. There could a possibility that Reichardt’s dye used in this study was impure. The dichloro- substituted form of

Reichardt’s dye, which has a lower pKa than the dye used in the present study, might also be used to counter the acidity of the solvent.

ET(30) literature value for (-)-Ethyl L-Lactate at T=20 °C was measured to be 51.1

N kcal/mol [8]. It was also found that the normalized parameter, ET decreases with temperature, especially above 40°C [3]. Based on the results obtained in this study, as shown in table 9,

ET(30 value of (-)-Ethyl L-Lactate is 57.30 kcal/mol at 25°C and decreases with temperature, which is consistent with the data in literature.

The Kamlet-Taft hydrogen bond acceptance parameter, , was calculated from absorbance wavelengths using equation 12 and is given in Table 10 and Figure 14.

Table 10: Hydrogen-Bond Acceptance (Basicity) for (-)-Ethyl L-Lactate, (+)-Ethyl D-Lactate and Racemic Mixture at T=10-70 oC β for (-)-Ethyl L- β for (+)-Ethyl D- β for Racemic T (K) Lactate Lactate Mixture 283 0.4958 0.3079 0.1077 298 0.4893 0.3605 0.0913 313 0.3725 0.2947 -0.0638 328 0.1926 0.1424 -0.1768 343 0.0689 0.1316 -0.1985

0.6

0.5

0.4

0.3

0.2

0.1β

0 (-)Ethyl-L-Lactate -0.1 (+)Ethyl-D-Lactate -0.2 Ethyl-D,L-Lactate -0.3 250 270 290 310 330 350 T (K)

Figure 14: Plot Showing Temperature Dependence of HBA (β) for (-)-Ethyl L-Lactate, (+)-Ethyl D-Lactate and Racemic Ethyl-D,L-Lactate Mixture

The HBA (β) trend for the three solvents in figure 14 is consistent with the literature [21].

Elevated temperatures affect the hydrogen-bond acceptance (basicity) of the solvent. Hydrogen bonding typically decreases with increases in temperature due to the higher thermal (kinetic) energy of the molecules. In figure 14, β trend for racemic ethyl lactate mixture shows a lower value in its hydrogen bond acceptance ability at all temperatures than either enantiomer. This suggests that presence of both enantiomers in the racemate are blocking access to the carbonyl oxygens.

The Kamlet-Taft hydrogen bond donor parameter, , was calculated from absorbance wavelengths using equation 10 and is given in Table 11 and Figure 15.

Table 11: Hydrogen-Bond Donation (Acidity) for (-)-Ethyl L-Lactate, (+)-Ethyl D-Lactate and Racemic Mixture at T=10-70 oC

α for Racemic T (K) α for (-)-Ethyl L-Lactate α (+)-Ethyl D-Lactate Mixture 283 1.0568 1.1690 1.0923 298 1.0723 1.1619 1.1304 313 1.0764 1.2021 1.1412 328 1.0867 1.2012 1.1679 343 1.0976 1.2176 1.2193

1.24

1.22 (-)Ethyl-L-Lactate

1.2 (+)Ethyl-D-Lactate

1.18 Ethyl-D,L-Lactate

1.16

1.14 α 1.12

1.1

1.08

1.06

1.04 250 270 290 310 330 350 T (K) Figure 15: Plot Showing Temperature Dependence of HBD (α) for (-)-Ethyl L-Lactate, (+)-Ethyl D-Lactate and Racemic Ethyl-D,L-Lactate Mixture

The HBD acidity (α) is the ability of a solvent to make hydrogen bond with a solute by donating its proton. From a theoretical stand point, both Kamlet-Taft parameters: HBD (α) and

HBA (β) decrease as the temperature increases [22]. α values of those three solvents as a function of temperature are shown in figure 15, which indicate that hydrogen-bond donor ability

(acidity) of those solvents is more favorable at elevated temperatures. One possible explanation for this unusual behavior is proton stabilization in the solvent due to intramolecular hydrogen bonding between the hydroxyl and carbonyl groups. The subsequent resonance stabilization that would result can also cause steric hindrance between enantiomeric pairs, leading to reduced access to the carbonyl oxygens and lower the value of , as observed in the previous discussion. It is also possible that the error in ET(30) values led to errors in the values for . Measurement of the ET(33) value using the dichloro- substituted Betaine’s dye, which was beyond the scope of this study, could help resolve this uncertainty.

Finally, the Kamlet-Taft polarization parameter, , was calculated from absorbance wavelengths using equation 9 and is given in Table 12 and Figure 16.

Table 12: Polarizability for (-)-Ethyl L-Lactate, (+)-Ethyl D-Lactate and Racemic Mixture at T=10-70 oC

π* for (-)-Ethyl L- π* for (+) Ethyl D- π* for Racemic T (K) Lactate Lactate Mixture 283 0.7326 0.6111 0.7326 298 0.6925 0.5905 0.6723 313 0.6520 0.5490 0.6520 328 0.6111 0.5280 0.5905 343 0.5905 0.4858 0.5280

0.75

0.7

0.65

0.6 π* 0.55

0.5 (-)Ethyl-L-Lactate (+)Ethyl-D-Lactate 0.45 Ethyl-D,L-Lactate 0.4 250 270 290 310 330 350 T (K)

Figure 16: Plot Showing Temperature Dependence of Polarizability (π*) for (-)-Ethyl L-Lactate, (+)-Ethyl D-Lactate and Racemic Ethyl-D,L-Lactate Mixture

The polarization/dipolarity (π*) data trend for the three solvents shown in figure 16 is consistent with the literature [21]. Figure 16 shows that π* values for those solvents in figure 16 decrease as the temperature increases. At higher temperatures, molecular motion increases, which makes it more difficult to align a dipole or hold a charge. The trend in figure 16 also suggests that π* of racemic mixture decreases at a faster rate than the D and L-form. Since π* values are related to van der Walls interaction and dielectric constant of a molecule, π* values of

41 the solvents indicate the dielectric constant is more sensitive to enantiomeric impurities at higher temperatures.

CONCLUSIONS Physical and solvent properties of enantiomeric mixtures of Ethyl lactate were measured and reported for the first time. The melting point of (-)-Ethyl L-Lactate in Material Safety Data

Sheet (MSDS) was found to be inaccurately documented. It is likely that the melting point (Tm= -

26 °C) belongs to the melting point of racemic mixture. Modulated DSC was also used to construct the binary phase diagram of ethyl lactate enantiomers. Based on the modulated DSC phase diagram, Tm of (-)-Ethyl L-Lactate was -6.22 °C . Melting point data on (+)-Ethyl D-Lactate was entirely absent in the MSDS, but this study measured it to be -4.23 °C. Binary phase diagram of ethyl lactate enantiomers also showed the existence of a solid – solid transition at around -40 °C. At this value, the crystal structure of the racemate changes. Phase diagram of binary enantiomeric mixture of ethyl lactate also showed a eutectic point for an eutectic

o composition of 60:40 [(L-form)/ (D-form)] at eutectic temperature Te= -26.95 C.

Specific rotation, [α] for (-)-Ethyl L-Lactate was -9.915 degrees, which agrees well with its literature documented [α] value of -10.5 degrees. The experimental [α] for (+)-Ethyl D-Lactate was +11.414. Polarimeter measurements indicated the possibility of an excess amount of (+)-

Ethyl D-Lactate in the pure L-sample. GC-MS revealed that there were more impurities in the

(+) Ethyl D-Lactate, and that it was likely that the impurities were optically active. The presence of lactate esters of higher molecular weight alcohols or ethyl 2-hydroxybutyrate could explain the higher specific rotations observed for the D-form ethyl lactate. Density and refractive index values at room temperature (20 °C) were within experimental error and that the refractive index and density were not influenced by the chirality or enantiomeric excess of the solvent.

Since solvent polarity is directly related to chemical kinetics and spectral absorption, this study used three traditional solvatochromic probe dyes: Reichardt’s Betaine Dye, N,N-Diethyl-4-

Nitroaniline and 4-Nitroaniline to measure polarity, hydrogen-bond donor ability, and hydrogen- bond acceptance ability of L-form, D-form and racemic mixture of ethyl lactate solvents. The

43 electronic transition maxima of those three solvatochromic probes in three solvents: (-)-Ethyl L-

Lactate, (+)-Ethyl D-Lactate and racemic ethyl lactate mixtures were measured using UV-Vis spectroscopy at T = 10-70 °C. The Reichardt’s polarity parameter, ET(30), did not show much difference in any solvent or at any temperature. The low absorbance values and acidic nature of the solvent indicate that the dye was protonated, losing its ability for intramolecular charge transfer. Use of a dye with lower pKa may resolve this issue.

The dipolarity/polarization of all three solvents decreased at higher temperature, which is consistent with the decrease in dielectric constant with increasing temperature observed in most solvents. At higher temperatures, hydrogen-bond donor (acidity) increased for (-)-Ethyl L-

Lactate, (+)-Ethyl D-Lactate and racemic mixture which concluded that hydrogen bond donation ability of three solvents became stronger at higher temperature. Hydrogen-bond acceptance

(basicity) for all three solvents decreased at higher temperature. It was also observed that the solvent parameters of the racemic mixture was an average of those of two pure enantiomers, with the exception of the hydrogen bond accepting parameter, . Strong intramolecular bonding between the hydroxyl and carbonyl groups could help explain this unusual behavior.

APPENDIX A: GC-MS RESULTS

Gas-Chromatography-Mass Spectrometry (GC-MS) Results: Chromatograms and Percent Purity for (-)-Ethyl L-Lactate, (+)-Ethyl D-Lactate, and Racemic Mixture. The gas chromatograms for (-)-Ethyl-L-Lactate, (+)-Ethyl D-Lactate, and the racemic mixture are shown in figures 17, 18, and 19. The MS spectrum for the first peak of all chromatographs was the same and is represented in Figure 20. The MS spectra for the second and third peaks of the D-form ester are shown in Figures 16 and 17. The NIST library searches for identifying the compounds present in each peak for (-)-Ethyl-L-Lactate, (+)-Ethyl D-Lactate, and the racemic mixture are provided in Figures 23, 24, and 25, respectively.

Abundance

TIC: 15-Mobeen.D\ data.ms 4000000

3500000

3000000

2500000

2000000

1500000

1000000

500000

0 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 Time--> Figure 17: GC Chromatogram of (-)-Ethyl L-Lactate

Abundance

TIC: 14-Mobeen.D\ data.ms 4000000

3500000

3000000

2500000

2000000

1500000

1000000

500000

0 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 Time--> Figure 18: GC Chromatogram of (+)-Ethyl D-Lactate

Abundance

TIC: 13-Mobeen.D\ data.ms

3500000

3000000

2500000

2000000

1500000

1000000

500000

0 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 Time--> Figure 19: GC Chromatogram of Racemic Ethyl Lactate Mixture

Ethanol

Acetic Anhydride

Figure 20: Mass Spectrum of the Main Peak in all Ethyl Lactate Solvents

Figure 21: Mass Spectrum of the 2nd peak in (+)-Ethyl-D-Lactate Solvent

Figure 22: Mass Spectrum of the 3rd peak in (+)-Ethyl-D-Lactate Solvent

Figure 23: GC-MS Summary displaying the Percent Purity of (-)-Ethyl L-Lactate

Figure 24: GC-MS Summary displaying the Percent Purity of (+)-Ethyl D-Lactate

Figure 25: GC-MS Summary Displaying the Percent Purity of Racemic Ethyl Lactate

APPENDIX B: MODULATED DSC THERMOGRAMS

100L/0D 90L/10D 80L/20D 70L/30D 60L/40D 50L/50D 40L/60D 30L/70D 20L/80D 10L/90D 0L/100D 45.000000

40.000000

35.000000

30.000000

25.000000

20.000000

15.000000 Heat Flow (mW)Flow Heat 10.000000

5.000000

0.000000 -70.000000 -60.000000 -50.000000 -40.000000 -30.000000 -20.000000 -10.000000 0.000000 10.000000 20.000000 -5.000000 T (°C) Figure 26: Showing DSC Cooling Curves for Ethyl Lactate Enantiomeric Mixtures obtained at a Heating Rate of 20 oC/min

DSC Thermogram for (-)-Ethyl L-Lactate:

Figure 27. DSC Thermogram showing the Melting Point and Enthalpy of Fusion of (-)-Ethyl L- Lactate Solvent

DSC Thermogram for (+)-Ethyl D-Lactate

Figure 28. DSC Thermogram showing the Melting Point and Enthalpy of Fusion of (+)-Ethyl D- Lactate Solvent

DSC Thermogram for Racemate (50% L-form/ 50% D-form)

Figure 29. DSC Thermogram showing the Melting Point and Enthalpy of Fusion of Racemic Ethyl Lactate Solvent

DSC Thermogram for Eutectic Mixture (60% L-form/40% D-form)

Figure 30. DSC Thermogram showing the Melting Point and Enthalpy of Fusion at Eutectic Point composition: 60:40 [(L-form)/(D-form] of Ethyl Lactate Solvent

APPENDIX C: ABSORPTION SPECTRA

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2

Abs ( A.U.) ( Abs 0.1 0 200 300 400 500 600 700 800 λ (nm)

A at T=10 °C A at T=25 °C A at T=40 °C A at T=55 °C A at T=70°C

Figure 31. Electronic Absorption Spectrum of Reichardt’s dye in (-)-Ethyl L-Lactate at T= 10, 25, 40, 55 and 70 oC

0.8

0.6

0.4

0.2

Abs.( A.U.) A.U.) Abs.( 0 200 300 400 500 600 700 800 λ (nm)

A at T=10 deg C A at T=25 deg C A at T= 40 deg C A at T=55 deg C A at T=70 deg C

Figure 32. Electronic Absorption Spectrum of Reichardt’s dye in (+)-Ethyl D-Lactate at T= 10, 25, 40, 55 and 70 oC

1 0.9 0.8 0.7 0.6 0.5 0.4

Abs ( A.U.) A.U.) ( Abs 0.3 0.2 0.1 0 200 300 400 500 600 700 800 λ (nm)

A at T=10 deg C A at T=25 deg C A at T= 40 deg C A at T=55 deg C A at T=70 deg C

Figure 33. Electronic Absorption Spectrum of Reichardt’s Dye in Racemic Ethyl Lactate Mixture at T= 10, 25, 40, 55 and 70 oC

2 1.8 1.6 1.4 1.2 1 0.8

Abs (A.U.) Abs 0.6 0.4 0.2 0 200 300 400 500 600 700 800 λ (nm)

A at T=10 oC A at T=25 oC A at T= 40 oC A at T=55 oC A at T=70 oC

Figure 34. Electronic Absorption Spectrum of DENA dye in (-)-Ethyl L-lactate at T= 10, 25, 40, 55 and 70 oC

2.5

1.5

Abs. (A.U.) Abs. 1

0.5

0 200 300 400 500 600 700 800 λ (nm)

A at T=10 °C A at T=25 °C A at T=40 °C A at T=55 °C A at T=70 °C

Figure 35. Electronic Absorption Spectrum of DENA dye in (+)-Ethyl D-lactate at T= 10, 25, 40, 55 and 70 oC

2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

200 300 400 500 600 700 800 Abs. (A.U.) Abs. λ (nm)

A at T=10 deg C A at T=25 deg C A at T= 40 deg C A at T=55 deg C A at T=70 deg C

Figure 36. Electronic Absorption Spectrum of DENA Dye in Racemic Ethyl Lactate Mixture at T= 10, 25, 40, 55 and 70 oC

2 1.8 1.6 1.4 1.2 1 0.8

Abs (A.U.) Abs 0.6 0.4 0.2 0 200 300 400 500 600 700 800 λ (nm)

A at T=10 oC A at T=25 oC A at T= 40 oC A at T=55 oC A at T=70 oC

Figure 37 . Electronic Absorption Spectrum of PNA dye in (-)-Ethyl L-lactate Solvent at T= 10, 25, 40, 55 and 70 oC

2 1.8 1.6 1.4 1.2 1 0.8

Abs(, A.U.) A.U.) Abs(, 0.6 0.4 0.2 0 200 300 400 500 600 700 800 λ (nm)

A at T=10 deg C A at T=25 deg A at T=25 deg C A at T= 40 deg C A at T=55 deg C A at T=70 deg C

Figure 38. Electronic Absorption Spectrum of PNA dye in (+)-Ethyl D-Lactate at T= 10, 25, 40, 55 and 70 oC

2 1.8 1.6 1.4 1.2 1 0.8

Abs( A.U. ) A.U. Abs( 0.6 0.4 0.2 0 200 300 400 500 600 700 800 λ (nm)

A at T=10 deg C A at T=25 deg C A at T= 40 deg C A at T=55 deg C A at T=70 deg C

Figure 39. Electronic Absorption Spectrum of PNA dye in Racemic Ethyl Lactate Mixture at T= 10, 25, 40, 55 and 70 oC

Table 13: Reichardt’s and Kamlet-Taft Parameter Results for (-)-Ethyl L-Lactate:

T (°C) Betaine DENA NA ET(30) Kcal/mol T (K) π* β α 10 498 397 366 57.4116 283 0.7325843 0.4958 1.0568 25 499 395 364 57.2966 298 0.6925029 0.4893 1.0723 40 502 393 358 56.9542 313 0.6520136 0.3725 1.0764 55 504 391 350 56.7282 328 0.6111101 0.1926 1.0867 70 504 390 345 56.7282 343 0.5905011 0.0689 1.0976 Table 14: Generated Reichardt’s and Kamlet-Taft Parameter Results for (+)-Ethyl D-Lactate:

T (°C) Betaine DENA pNA ET(30) Kcal/mol T (K) π* β α 10 490 391 354 58.3490 283 0.6111101 0.3079 1.1690 25 493 390 355 57.9939 298 0.5905011 0.3605 1.1619 40 490 388 351 58.3490 313 0.5489642 0.2947 1.2021 55 492 387 345 58.1118 328 0.5280348 0.1424 1.2012 70 493 385 343 57.9939 343 0.4858498 0.1316 1.2176

Table 15: Reichardt’s and Kamlet-Taft Parameter Results for Racemic Ethyl Lactate Mixture:

T (°C) Betaine DENA pNA ET(30) Kcal/mol T (K) π* β α 10 492 397 352 58.1118 283 0.7325843 0.1077 1.0923 25 491 394 349 58.2301 298 0.6723097 0.0913 1.1304 40 491 393 343 58.2301 313 0.6520136 -0.0638 1.1412 55 492 390 337 58.1118 328 0.5905011 -0.1768 1.1679 70 489 387 334 58.4683 343 0.5280348 -0.1985 1.2193

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