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Thermodynamic Study of Enantiomeric Separations Using
Normal Phase Chiral HPLCGregory Perkins
Senior ThesisSpring 2009
Advisor: Debra L. Van Engelen
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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
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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.
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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.
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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.
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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.
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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.
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Chromatography
ResolutionR = tr / w
Christian, Gary. Analytical Chemistry, 5th Edition, John Wiley and Sons, New York 1994.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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
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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
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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
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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
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Instrumentation Shimadzu LC-20ACT solvent delivery systems
with dual reciprocating plunger design Shimadzu SPD-20AV HPLC UV-Vis Detector
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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
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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
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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
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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)
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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
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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
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Van’t Hoff Plot of Chiralpak IA column separation of flavanone enantiomers at 4 temperatures, 4 replicates each with hexane and isopropanol mobile phase
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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
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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)
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FlavanoneThermodynamic Analysis Slope = -H/R = 51.723
H = -430 J
Y-intercept = S/R = -0.0685S = -0.570 J/K
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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
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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