Greg Perkins SR THESIS PRES version 4

Thermodynamic Study of Enantiomeric Separations Using

Normal Phase Chiral HPLCGregory Perkins

Senior ThesisSpring 2009

Advisor: Debra L. Van Engelen

Introduction

Pharmaceutical industryR and S have enantiomers different

propertiesFDA prefers single enantiomers

>1/2 of Top 500 drugs

Single-enantiomer drug sales increases every year

Expanding field of business

Non-Separation Techniques

Polarimetry Circular dichroism Nuclear Magnetic Resonance using

shift agents Enantioselective sensors

– Enzyme reactions

Christian, Gary. Analytical Chemistry, 5th Edition, John Wiley and Sons, New York 1994.

Other Separation Techniques

Synthesize diastereoisomers & separate on achiral column– HPLC on C-18 column

CEC - Capillary electrochromatography– Add an pseudo-stationary phase enantiomer to a

buffer Direct separation by high performance liquid

chromatography (HPLC)– Chiral Stationary Phase (CSP)

Christian, Gary. Analytical Chemistry, 5th Edition, John Wiley and Sons, New York 1994.

Brush Type Columns

Creator: William Pirkle (1970’s)Low molecular weight selectorChiral selector molecules act independentlyAttractive Interactions

Pi-bonding H-bondingVan der WaalsDipole interactions

Separate large # of pharmaceuticalsYueqi, Liu; Wenjian, Lao; Yuhua, Zhang; Shengxiang, Jiang; Liren, Chen. 泥 irect Optical Resolution of the enantiomers of axially chiral compounds by high-performace liquid chromotography on cellulose tris-(3,5-dimethyl phenylcarbamate) stationary pahses, Chromotographia, 2000, 52, 190-194.

Polysaccharide Columns Cellulose or Amylose based Contain chains that form sheets

– Inclusion Complexes & Attractive Interactions• Cavities in carbohydrate chain

Derivitization increases enantioselective properties

Chiral recognition based on:– Shape– H-bonding– dipole-dipole - interactions

Yueqi, Liu; Wenjian, Lao; Yuhua, Zhang; Shengxiang, Jiang; Liren, Chen. “Direct Optical Resolution of the enantiomers of axially chiral compounds by high-performace liquid chromotography on cellulose tris-(3,5-dimethyl phenylcarbamate) stationary pahses,” Chromotographia, 52, 2000, 190-194.

Chiralpak IA Bonded stationary phase can

use a wider range of solvents Carbohydrate Type Column

– Normal phase only– Mobile Phase

• Mixture of hexane & alcohol Chiral Selector

– Amylose tris(3,5-dimethylphenylcarbamate) Solid Support Diameter

– 5 m

Chiralpak IA. Chiral Technologies Product Literature, West Chester, PA.

Chromatography

ResolutionR = tr / w

Christian, Gary. Analytical Chemistry, 5th Edition, John Wiley and Sons, New York 1994.

Quality of Separation

Number Theoretical PlatesN = 16 (tr /w)2

Capacity Factork’ = (tr - tm) / tm

Selectivity Factor = k 'B / k 'A

Christian, Gary. Analytical Chemistry, 5th Edition, John Wiley and Sons, New York 1994.

Thermodynamics of Chromatography

Distribution coefficientKD = Cs / Cm

Cs =concentration of solute in stationary phase

Cm=concentration of solute in mobile phase

Capacity factork’ = (tr - tm) / tm

k’ = KD (Vs / Vm) = KD =phase ratio or (Vs / Vm)

[4] Schrum, D. The Synthesis and Characterization of Polyacrylate Anion-Exchange Stationary Phases for Protein Separations in Liquid Chromatography (Thesis), Purdue University Graduate School: 1996, 125-160.

Thermodynamic Properties

At equilibrium,G = - RT ln KD

Substituting and rearrangingG = - RT ln k’ (Vm/Vs) G = -RT ln k’ (1/) G = - RT [ln k’ + ln (1/)]

G = - RT [ln k’ - ln ] [4] Schrum, D. The Synthesis and Characterization of Polyacrylate Anion-Exchange Stationary Phases for Protein Separations in Liquid Chromatography (Thesis), Purdue University Graduate School: 1996, 125-160.

Traditional Van’t Hoff Plots in ChromatographySubstituting G = H -TS H -TS = - RT [ln k’ - ln ] Rearrangeln k’ = -H / RT + S / R + ln If is known and constant, a plot of ln k’vs 1/T

Slope of -H / RT Y-intercept of + S / R + ln

[4] Schrum, D. The Synthesis and Characterization of Polyacrylate Anion-Exchange Stationary Phases for Protein Separations in Liquid Chromatography (Thesis), Purdue University Graduate School: 1996, 125-160.

Polysaccharide CSP for HPLC unknown because volumes change

with mobile phase conditions Adapted Van’t Hoff plot

– Where G, H and S are the differences of G, H and S for a given pair of R and S enantiomers

– Temperature will be changed– Mobile phase composition will remain

constant

Weng, W; Zeng, Q; Yao, B; Wang, Q; Li, S. Chromatographia. 2005, 61, 561-566.

Adapted Van’t Hoff Plot

The change in free energy will be reflected in the relative capacity factors for each enantiomer

G = -RT ln (kr’/ks’)Where is constant and drops out for both

enantiomersSubstituting:

ln = -H / RT + S / RPlot of ln vs 1/T

Slope = -H / R Intercept = S / R

Weng, W; Zeng, Q; Yao, B; Wang, Q; Li, S. Chromatographia. 2005, 61, 561-566.

Previous Research Weng et al: Polysaccharide column separating

derivatized amino acids and binapthyl compounds

Hexane and 2-propanol– <3% alcohol had lower tr and better separation

Hydrogen bonding & π- π interactions– AA w/ phenyl had larger enantioselectivity than

methyl

Weng, W; Zeng, Q; Yao, B; Wang, Q; Li, S. Chromatographia. 2005, 61, 561-566.

Weng et al (continued) AA separated at different temperatures

– ln vs. 1/T– Enthalpy driven

H and S both negative

Weng, W; Zeng, Q; Yao, B; Wang, Q; Li, S. Chromatographia. 2005, 61, 561-566.

Amino Acid Derivative

Enthalpy driven– Strong H-bonding or - interactions w/ CSP– Phenyl group enhanced– Methyl group has less interactions

% 2-Propanol 25 C 30 C 35 C 40 C

H (kJ / ol)

S (JK/ o l)

12 1. 6 9 1. 6 3 1. 5 6 1. 5 0 - 6. 4 1 - 17 . 11

9 1. 7 1 1. 6 4 1. 5 7 1. 5 0 - 6. 5 3 - 17 . 42

6 1. 7 2 1. 6 5 1. 5 8 1. 5 1 - 6. 4 9 - 17 . 23

3 1. 7 6 1. 6 8 1. 6 1 1. 5 4 - 6. 7 5 - 17 . 95

1.5 1. 8 6 1. 7 8 1. 7 2 1. 6 7 - 5. 5 1 - 13 . 37

1 1. 9 8 1. 9 0 1. 8 6 1. 8 2 - 4. 4 1 -9. 1 2

F

COOMe

NHCOPh

Binapthyl Compounds

More Entropy Driven– Steric Effects

OH

OH

% 2-Propanol 25 C 30 C 35 C 40 C

H° (kJ /m o l)

S° (J / Km o l)

12 1. 1 8 0 6 1. 1 7 5 8 1. 1 7 3 9 1. 1 6 9 6 - 0. 4 6 - 0. 1 5

9 1. 1 8 7 0 1. 1 8 4 7 1. 1 7 9 7 1. 1 7 5 9 - 0. 5 0 - 0. 2 5

6 1. 1 8 9 5 1. 1 8 7 2 1. 1 8 3 9 1. 1 7 9 6 - 0. 4 3 0. 0 1

3 1. 1 9 3 6 1. 1 9 1 1 1. 1 8 8 4 1. 1 8 5 0 - 0. 3 7 0. 2 2

My Research Determine separations of enantiomers

on Chiralpak IA column are enthalpy or entropy driven

3-hydroxypropranoic acid-1-phenyl-ethylester (3HP-1EE) and Flavanone

O OH

O O

O

Experimental 3HP-1EE & Flavanone

– Chiralpak IA• Polysaccharide Column

– Normal Phase• Hexane and Isopropanol• Varying %, 95/5, 90/10, 85/15

Vary Temperature– 20-35 C (water bath)

15 minute run times

Instrumentation Shimadzu LC-20ACT solvent delivery systems

with dual reciprocating plunger design Shimadzu SPD-20AV HPLC UV-Vis Detector

Separations of 3HP-1EE

Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

20

40

60

mAU

0

20

40

60

3.117

45040

3.700

2286

3.850

2065

4.017

5700

4.775

5603

4.950

1450

5.158

1481

5.550

6670

5.900

622

6.425

575594

6.992

5710577.950

6175

SPD-20AV Ch 1-254nmso u lsby25C11-3-2008 2-22-31 PMso u lsb y25C.dat

Retention TimeArea

Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

20

40

60

mAU

0

20

40

60

3.150

46702

3.717

2321

3.858

1921

4.008

5761

4.742

5426

4.925

1071

5.092

837

5.533

5520

6.333

581991

6.842

623108 7.642

18651

7.958

39196

8.258

79131

9.200

65037

SPD-20AV Ch 1-254nmso u lsby30C11-3-2008 2-58-04 PMso u lsb y30C.dat

Retention TimeArea

Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

20

40

60

mAU

0

20

40

60

3.158

52725

3.942

9001

4.108

1972

4.650

5732

4.825

975

4.975

811

5.192

882

5.458

5499

6.158

620006

6.600

6349667.442

1379

SPD-20AV Ch 1-254n msoulsb y35C11-3-2008 4-04-54 PMsou lsb y35C.dat

Retention TimeArea

Van’t Hoff Plot of Chiralpak IA column separation of 3HP-1EE enantiomers at 4 temperatures, 4 replicates each with hexane and isopropanol mobile phase

3-hydroxypropranoic acid-1-phenyl-ethylester (3HP-1EE)

ln a0.1380.1310.1260.1220.1160.108

1/T0.0034130.0033840.0033560.0033280.0032790.003247

t t1 t2 w1 w2 k'1 k'2 R N T 3.29 10.46 12.00 0.53 0.58 1.15 2.18 2.65 2.79 6230 20.0 3.27 10.25 11.68 0.51 0.57 1.14 2.13 2.57 2.68 6600 22.5 3.28 10.05 11.40 0.50 0.55 1.13 2.06 2.48 2.59 6620 25.0 3.27 9.92 11.21 0.52 0.54 1.13 2.03 2.43 2.42 5830 27.5 3.24 9.69 10.88 0.50 0.54 1.12 1.99 2.36 2.30 6140 32.0

3.278 10.01 11.34 0.51 0.55 1.114 1.93 2.26 2.13 6210 35.0

3HP-1EE y = 171.61x - 0.4487R2 = 0.9899

0.100

0.105

0.110

0.115

0.120

0.125

0.130

0.135

0.140

0.0032 0.00325 0.0033 0.00335 0.0034 0.003451/T

ln a

Van’t Hoff Plot of Chiralpak IA column separation of 3HP-1EE enantiomers at 6 temperatures, 2 replicates each with hexane and isopropanol mobile phase (95/5)

3-hydroxypropranoic acid-1-phenyl-ethylester (3HP-1EE) Thermodynamic Analysis

Slope = -H/R = 171.61 H = -1430 J

Y-intercept = S/R = -0.4487S = -3.73 J/K

Separations of Flavanone (95/5) with Increasing Temperature

Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

50

100

150

mAU

0

50

100

150

6.558

32763

7.867

1325473

8.642

1541472

9.817

1666

SPD-20AV Ch1-254n mflavan o n e21C9-26-2008 1-19-06 PMflavano n e21C.d at

Retention TimeArea

Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

200

400

mAU

0

200

400

6.342

83886

7.433

3579008

8.117

3623691

9.283

4056

9.592

2660

SPD-20AV Ch1-254n mflavan o n e30C9-26-2008 2-35-40 PMflavano n e30C.d at

Retention TimeArea

Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

200

400

mAU

0

200

400

6.225

73329

7.200

3644615

7.858

3692804

9.050

6986

SPD-20AV Ch1-254n mflavan o n e35C9-26-2008 3-32-47 PMflavano n e35C.d at

Retention TimeArea

Van’t Hoff Plot of Chiralpak IA column separation of flavanone enantiomers at 4 temperatures, 4 replicates each with hexane and isopropanol mobile phase

Flavanone

ln a0.1080.1070.1040.1040.1020.1010.100

1/T0.003400.003380.003360.003330.003300.003280.00325

t t1 t2 w1 w2 k'1 k'2 R N T 3.325 7.861 8.756 0.33 0.35 1.114 1.364 1.633 2.650 9147 21 3.329 7.813 8.692 0.30 0.33 1.113 1.347 1.611 2.819 11308 23 3.329 7.700 8.546 0.28 0.29 1.110 1.313 1.567 2.993 12557 25 3.329 7.642 8.479 0.30 0.31 1.110 1.296 1.547 2.767 10746 27 3.325 7.504 8.313 0.27 0.30 1.108 1.257 1.500 2.863 12359 30 3.333 7.438 8.224 0.27 0.27 1.106 1.231 1.467 2.943 12616 32 3.338 7.325 8.096 0.26 0.27 1.105 1.195 1.426 2.965 13215 35

Flavanone 95/5 y = 51.723x - 0.0685R2 = 0.9656

0.0990.1000.1010.1020.1030.1040.1050.1060.1070.1080.109

0.00320 0.00325 0.00330 0.00335 0.00340 0.003451/T

ln a

Van’t Hoff Plot of Chiralpak IA column separation of flavanone enantiomers at 7 temperatures, 2 replicates each with hexane and isopropanol mobile phase (95/5)

FlavanoneThermodynamic Analysis Slope = -H/R = 51.723

H = -430 J

Y-intercept = S/R = -0.0685S = -0.570 J/K

Summary & Conclusions 3HP-1EE

– As temperature increases…• Retention time and capacity factor decrease• Resolution decreases while theoretical plates is

constant• Selectivity factor decrease

– Enthalpy Driven• -H large• Entropy is not favorable

O OH

O

Summary & Conclusions

Flavanone– As temperature increases…

• Retention time and capacity factor decrease• Selectivity factor decreases• Resolution and theoretical plates increase

– Somewhat Entropy Driven ?• very small -S (and -H)• Steric effects contribute

O

O