Senior Thesis FINAL 1

Thermodynamic Study of Enantiomeric Separations Using Normal Phase Chiral HPLC

Perkins, Gregory

University of Redlands: Senior ThesisSpring 2009

Abstract:

Normal Phase Chiral HPLC was used to determine if 3-hydroxypropranoic acid-

1-phenyl-ethylester (3HP-1EE) and flavanone enantiomeric separations are enthalpy or

entropy driven. A Chiralpak-IA polysaccharide column was used to separate the two

compounds and temperature was controlled by a water bath. Both compounds were

tested at various temperatures with multiple runs at each temperature. A Van’t Hoff plot

graphing ln vs 1/T (1/K) was used to obtain S and H values for both

compounds. 3HP-1EE had a H = -1430 J and a S = -3.73 J/K, showing that is

primarily enthalpy driven. Flavanone had a H = -430 J and a S = -0.570 J/K,

showing that it is more entropy driven. This means that larger compounds will be

entropy driven and easily separated at higher temperatures while smaller compounds will

be enthalpy driven and easily separated at lower temperatures.

Introduction:

Separation of pharmaceutical enantiomers is of great importance to the

pharmaceutical industry. Individual enantiomers can exhibit different pharmaceutical,

toxicological, metabolic, and pharmacokinetic properties within chiral environments or

they may react at different rates. The U.S. Food and Drug Administration (FDA) prefer

single enantiomers whenever it is practical and economically feasible. This is because

different enantiomers of the same molecule can have different effects in the body.

1

Today, more than half of the top 500 drugs are single enantiomers. An example of a

single enantiomeric drug is Lipitor, which has generated profits in the multi-billion dollar

range since its release.

Figure 1. A molecular diagram of Lipitor with two R* chiral centers where the OH groups are shown to have stereochemistry. < www.drugs.com/ pro/lipitor.html>

There are several methods for evaluating enantiomeric drug purity. Non-

separating techniques include polarimetry, circular dichroism, Nuclear Magnetic

Resonance (NMR) using shift reagents, enzyme reactions and the use of enantioselective

sensors [1]. Polarimetry and circular dichroism both use polarized light to detect

different enantiomers [1]. NMR with shift reagents is similar to NMR with reagents

added to create chemical shifts that simplify the data [2]. This is usually used to separate

resonance structures of complex molecules that generally overlap. Enzyme reactions use

stereoselective enzymes that are more attracted to a certain enantiomer [1]. The last non-

separation technique uses enantioselective sensors, which are selective to different

enantiomers of a compound.

There are also separation techniques that can be used on samples containing

racemic mixtures. The first separation method is to react each chiral compound with a

2

chiral enantiomer to create two diastereoisomers so they can be separated on a C-18

chiral column using high performance liquid chromatography (HPLC). The second

method is capillary electrochromatography, or CEC, which adds an enantiomer to the

electrophoresis buffer so that one enantiomer in the sample will interact more with

enantiomers in the running buffer to create a differences in electrophoretic mobilities for

each enantiomer.

Another separation technique that will be the main focus of this paper is direct

separation of enantiomers by passing a mixture through a chiral stationary phase HPLC

column. Samples are injected and passed through the column where one of the chiral

centers, either R* or S*, will be more attracted to E* in the column, were E* is the

bonded enantiomeric center on the stationary phase. This difference in interaction will

create a separation between the enantiomers. Two of the most commonly used chiral

stationary phases for HPLC are the polysaccharide type and the Brush, or Pirkle, type.

Polysaccharides are either cellulose or amylose based chains (see Figure 2) which

are held together by hydrogen bonding to form sheets [3]. These sheets are held together

by van der Waals forces and the three-dimensional formation can sometimes create

inclusion complexes. These inclusion complexes are in cavities created during the

bonding of the chains that will interact with enantiomers. Each polysaccharide sub-unit

will contain several chiral centers with chiral activities, but the interactions with

enantiomers are weak, so derivitization by a reaction at the hydroxyl group to add

additional chiral centers can improve enantioselective properties [3]. Recognition of

chiral compounds by the polysaccharide column are based on 3-D shape, chirality, H-

bonding, dipole interactions, pi interactions, and van der Waals forces.

3

Figure 2. An example of amylose chain with likages. (John Innes Centre, UK) <www.jic.ac.uk/STAFF/ cliff-hedley/Starch.html>

Brush-type columns, or Pirkle columns, consist of low molecular weight selectors

bonded to a silica substrate. A man named William Pirkle first introduced these columns

in the 70’s [3]. The design allows three bonded separate chiral selector molecules to act

independently with each solute enantiomer and is compatible with many mobile phase

solvents. The selector molecules are made up of amino acid derivatives that consist of

aromatic rings, creating pi-pi interactions with the solute [3]. Other attractive interactions

that will stabilize the complex are H-bonding, dipole interactions, van der Waals forces,

steric repulsion, and Lewis acid or Lewis base interactions.

4

Figure 3. A chiral selector from a Nucleosil Chiral-2 brush type column. This column will experience Lewis acid/base interactions, H-bonding, dipole-dipole interactions and steric effects. (Macherey-Nagel) < http://www.mn-net.com/HPLCStart/SpecialHPLCphases/HPLCenantiomer/NUCLEOSILCHIRAL/tabid/6150/language/en-US/Default.aspx>

To separate these enantiomers, high performance liquid chromatography,

or HPLC, is used and it can be performed in two ways. The most common type of

mobile phase used for HPLC is reverse phase, where the solvent is more polar than the

stationary phase. Often to examine chiral stationary phases use the normal phase

technique where the mobile phase is less polar than the stationary phase.

The quality of a separation using HPLC can be determined using several different

equations. The number of theoretical plates tells the separation efficiency for the two

enantiomers. The number of theoretical plates can be determined using equation 1.

N = 16 (tr / w)2 (1)

For this equation, N is the number of theoretical plates, tr is the retention time and

w the baseline width of the peak. The capacity factor (equation 2) is a measure of how

long the enantiomer is retained by the column. A larger capacity factor means the

enantiomer is retained longer.

k’ = (tr - tm) / tm (2)

5

For this equation, k’ is the capacity factor, which is the retention time, tr, minus the time

for mobile phase, tm, divided by the time for the mobile phase. Resolution is another term

that can determine the quality of a separation, which shows how well the peaks are

separated between peaks that can be solved using equation 3.

R = tr / w (3)

R is the resolution, which is the difference in the retention time for the two peaks over the

average baseline width of the peaks. The selectivity factor, , or relative retention,

shows the quality of separation between two enantiomers. The greater the selectivity

factor is, the better the separation will be.

= k 'B / k 'A (4)

Alpha the selectivity factor is k’ of species B over that of A, where B is always the

second peak eluded from the column.

Thermodynamic properties can also be determined from HPLC retention data by

determining the capacity factor, k’, and the phase ratio, . The capacity factor, as

described above, can be expressed in the following ways:

k’ = (tr – t0) / t0 (5)

k’ = time of solute in stationary phase/time of solute in mobile phase (6)

Vs is the volume of the stationary phase and Vm is the volume of the mobile phase. The

phase ratio, , is equal to Vs / Vm, which is in the volume of the stationary phase, Vs, over

the volume of the mobile phase, Vm. In protein and polysaccharide columns the phase

ratio is not constant due to changes in the shape and volume of the stationary phase with

the changes in the mobile phase composition.

k’ = KD (Vs / Vm) = KD (7)

6

KD = Cs / Cm (8)

k' is the time in the stationary phase divided by the time in the mobile phase. The

distribution coefficient, KD, for a chromatographic system is also affected by the

thermodynamics of a solution [4]. Cs and Cm are concentrations for the stationary and

mobile phases, respectively.

The Selectivity Coefficient, , reflects k’ even though chromatographic system

may not reach equilibrium so it may not be equal in value. From thermodynamics, at

equilibrium, the free energy, G, equals –RT ln k so

G = - RT ln KD (9)

where R is the gas constant, and T is the temperature in Kelvin, K. Substituting equation

7; we get:

G = - RT ln k’ (Vm/Vs) = -RT ln k’ (1/) (10)

Rearranging equation 10 we get

G = - RT [ln k’ + ln (1/)] (11)

G = - RT [ln k’ - ln ] (12)

Using equation 13

G = H -TS (13)

and substituting it into equation 12:

H -TS = - RT [ln k’ - ln ] (14)

Rearranging equation 14

ln k’ - ln = -H / RT + S / R (15)

7

When this equation is plotted, in what is called a Van’t Hoff plot, with ln k’ vs. 1/T it will

have an intercept of S / R and a slope of -H / R [4]. G, H, S are define as

the differences of G, H and S for a given pair of enantiomers, R and S [5].

Then

G = -RT ln (kr’/ks’) = -RT ln (16)

then

ln = -H/RT + S/R (17)

Since there is no change in in a single isocratic chromatogram, that is, there is no

change in solvent composition, then

(ln R / ln S) = ln 1 = 0 (18)

The H and S values were determined by using a Van ’t Hoff plot of the natural log

of the selectivity factor versus the inverse of Temperature, in Kelvin. The slope of the

line is solved using equation 15 [5], where R is the gas constant.

Slope = -H / R (19)

The intercept is then used to determine the S value [5], with R as the gas constant

once again.

Intercept = S / R (20)

Several thermodynamic studies using these equations have been done and are presented

below

Weng et al. researched how to avoid the trial and error in the selection of

enantioseparation method [5]. The experiment was done on a Kromasil CHI-DMB

Brush-type column, consisting of an immobilized network polymer, with four amino acid

derivatives and two binaphthyl compounds that were enantioseparated successfully under

8

normal conditions. This experiment showed the significance that hydrogen bonding that

takes place in the mobile phase with the amino acids. The mobile phase for this

experiment was a mixture of n-hexane with differing amounts of 2-propanol. The

retention times of tested chiral compounds decreased and the separation of the

enantiomers increased tremendously when the amount of 2-propanol was below 3%. The

experiment also showed that the separated enantiomers of amino acid derivatives have

higher values and better separation than the enantiomers of binaphthyl compounds [5].

Enantiomers of amino acids with a phenyl group also had a larger enantioselectivity than

amino acids derivatives with a methyl group.

F

COOMe

NHCOPh

OH

OH

Figure 4. Example of one Amino Acid Derivitive and Binapthyl compound used in the Weng et al. experiment.

From van’t Hoff plots of ln vs (1/T), Weng et al concluded that

enantioseparations for the amino acids are enthalpy driven [5]. This is because both

H and S values of all four amino acids derivatives were negative, showing that

H is favorable and S is unfavorable

9

Figure 5. Weng et al van’t Hoff plot analyzing the effects of different temperatures and alcohol concentrations. From the graph on the far right, H and S values can be obtained [5].

Strong hydrogen bonding interactions or - interactions occurs with the

enantiomer and the Chiral Stationary Phase, CSP, which are enthalpy driven. The phenyl

group will enhance interactions more than a CH3 group because of additional -

interactions between the aromatic ring of CSP and the benzene ring of the solute. In the

larger compounds the OH groups are vital for the chirality of the compound but do not

enhance chiral recognition.

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

H (kJ /m ol)

S (JK/ m ol)

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

Figure 6. Data for the Amino Acid derivative (pictured above) indicating it is largely enthalpy driven.

10

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

H° (kJ /m o l)

S° (J /K m 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

Figure 7. Data for the Binaphthyl compound (pictured above) indicating that it is more entropy driven compared to the amino acid derivative.

The binaphthyl compounds have a less negative H value than the amino acids

because of the size of the compound [5]. Looking closer at the binaphthyl compounds,

the small change in H and S values compared to the amino acid derivatives shows

that steric hindrance causes enantioselectivity, not covalent bonding. But with an

increase in temperature the binaphthyl compounds could be more easily separated

because they are more entropy driven. This may reflect a conformational change in the

CSP polysaccharide at higher temperature. The large binaphthyl compounds have

hydrogen bonding because of the OH groups but lack other strong interactions due to

steric hindrance [5]. The results of this experiment indicate that the size of the compound

may determine whether or not it is enthalpy or entropy driven.

Yueqi et al. tried to take a different approach in the study of Biphenyl dimethyl

dicarboxylate derivatives (DDB) with a chiral HPLC column consisting of a CSP of

cellulose tris-(3,5-dimethylphenylcarbamate). DDB is used to treat hepatitis and shows

the potential to act against HIV-1. For this experiment, the cellulose CSP column was

specially made by the researchers but prepared according to standard procedure. In this

experiment, various mixtures of hexane and several different alcohols were tested as the

11

mobile phase. Enantiomers of nineteen different compounds were separated tested under

these conditions including five carboxylic acids, eight -biphenyl dimethyl dicarboxylate

derivatives, and eight -biphenyl dimethyl dicarboxylate and derivatives. For most

compounds, the best mobile phase modifier, MPM, was 2-propanol and for four -DDB

and one -DDB the best MPM was 1-butanol. Little changes in values were observed,

but the k’ value did decrease as the concentration of alcohol increased. Along with

different alcohols, a change in amount of alcohol in the mobile phase was tested. The

results indicated that the alcohol that worked best with all compounds was 2-propanol

over other alcohols [6]. A change in temperature was also tested and it seemed that the

compounds were separated more efficiently (sharper peaks) at higher temperatures even

though enantioselectivity is decreased (less enantiomeric separation). These results are

shown in Figure 5. At higher temperatures, the separations will occur at a faster rate and

the peaks will be much higher and thinner than lower temperature separations. So from

this research, the researchers concluded that enantioselectivity is affected by temperature

and is entropy driven [6].

Figure 8. Data that shows the effect of Temperature on the selectivity factor, Resolution and capacity factor [6].

In Jacobson et al. the chiral stationary phase was immobilized bovine serum

albamin (BSA) on a solid support and this column was used to separate L- and D-isomers

of benzoyl derivatives of amino acids. The CSP uses long protein chains that allow BSA

12

to covalently bond to wide pore silica [7] which is similar to the polysaccharide column

used in the experiments mentioned . In this experiment, there were two different types of

interactions taking place in the stationary phase in the column; chiral interactions and

non-chiral interactions. There were two different chiral interactions that were taking

place between the amino acids and the column. The D-amino acid experienced a three-

point interaction with the column [8]. This three-point interaction is the strongest

possible interaction taking place because it sterically fits. This interaction was a high-

energy interaction that is more sensitive to temperature.

The second chiral interaction in the column, from Jacobson et al, was a two-point

interaction with the L-amino acid [8]. The chiral interactions on the column were easily

saturated because it had a limited number of sites, thus this interaction worked better at

lower concentrations of analyte. The other interactions taking place in the column were

non-chiral interactions. These were one-point interactions, van der Waals forces, polar

interactions and hydrogen bonding which were all equal for both D and L–amino acids.

The relative energies for the two separation modes were found using the enthalpies of

adsorption. At lower temperatures, non-chiral sites were most important and the system

was more ordered. At higher temperature, the system underwent a conformational

change, was less absorbed and became more chirally selective.

For this research plan, a set a similar shaped chiral compounds and test their

separations in a few different ways. Conditions were varied in the mobile phase and

tested with hexane and various alcohols at various concentrations. To further test

separations of the compounds temperatures were varied from 20-40 C. The affect of

entropy and enthalpy on chiral separations were accumulated. Van ‘t Hoff plots were

13

acquired to determine the H and S values for each separation. These methods

were tested on the Chiralpak IA, a polysaccharide column with Amylose tris(3,5-

dimethylphenylcarbamate) as its chiral selector.

Experimental:

The HPLC instrument was two Shimadzu LC-20ACT solvent delivery systems

with dual reciprocating plunger design. The Detector was a Shimadzu SPD-20AV HPLC

UV-Vis Detector. It has a large sensitivity level having a 0.5x10-5 AU max noise level

reading. Samples were injected into the instrument in 10 mL aliquots. The data was

observed on a Windows 98 based computer with an EZstart program.

The Chiralpak IA (Chiral Technologies Inc. and Daicel Chemical Industries,

LTD, West Chester, PA) is a carbohydrate type column that is used with normal mobile

phase. It’s chiral selector is Amylose tris(3,5-dimethylphenylcarbamate with a solid

support diameter of 5 mm. With this column the normal-phase mobile phase consisted of

hexane (HEX) and isopropanol (IPA) in ratios of 95/5, 90/10 and 85/15, respectively. All

mobile phase solvents and reagents were purchased from Aldrich, Milwaukee, WI unless

otherwise stated. Two chiral sample solutions were used during May 2008 which were 3-

hydroxypropranoic acid-1-phenyl-ethylester (3HP-1EE) and flavanone. 3HP-1EE was

400 ppm in IPA and synthesized by Dr. David Soulsby. Flavanone was 300 ppm in 90:

10 HEX: ethanol.

During Fall 2008 and Spring 2009 the compounds were tested to find if the

separations were more enthalpy or entropy driven. To do this a carbohydrate-type

column was placed in a water bath to control the temperature of the column. Each

compound was tested 3 times at a temperature ranging from 20-35C and varying alcohol

14

from 5-15 %. The samples tested were flavanone and 3HP-1EE, the same concentrations

as before. The separation was performed in the normal phase so hexane with isopropanol

was used as the mobile phase. Each Separation run time was about 15 minutes long and

the peaks came off between 5 and 10 minutes depending on conditions

O OH

O

O

O

Figure 9. The compound on the left is 3HP-1EE and Flavanone is pictured on the right.

Figure 10. Both possible chains, Amylose and Cellulose, with possible chiral selectors. The Chiralpak IA is amylose base using the selector on the right.

15

Data/Results:

Figure 11. Separations for 3HP-1EE in 95/5 conditions with temperatures at 23, 25, 28, 31 and 35 C from top left down and right.

16

Figure 12. Shows original data obtained for 3HP-1EE. The separations were done in replicates of 3 at 4 temperatures. 90/10 and 85/15 indicate the percent hexane to isopropanol ratio.

Data values for the figure above are shown in the appendix. The data collected

showed good correlation but it was than tested at 6 different temperatures with duplicates

of 2 runs. This new data was collected in Table 1.

t t1 t2 w1 w2 k'1 k'2 R N T3.29 10.46 12.00 0.53 0.58 1.15 2.18 2.65 2.79 6230 20.03.27 10.25 11.68 0.51 0.57 1.14 2.13 2.57 2.68 6600 22.53.28 10.05 11.40 0.50 0.55 1.13 2.06 2.48 2.59 6620 25.03.27 9.92 11.21 0.52 0.54 1.13 2.03 2.43 2.42 5830 27.53.24 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 35.0Table 1. Data for 3HP-1EE collectd at 6 temperatures at 95% Hexane, 5 % Isopropanol.

17

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

Figure 13. Van’t Hoff plot for 3HP-1EE to determine H and S values by using the ln vs. 1/T (1/K) This was using 95%hexane/5%isopropanol.

The slope of the line and y intercept is than used to fins the H and S

values. Using equation 19, the slope of 171.61 is used to determine the H, which is -

1430 J. With equation 20, the y-intercept is used to determine the S value, which is -

3.73 J/K.

The same method was than used on Flavanone.

18

Figure 14. Separations of falvanone under 95/5 conditions varying the temperatures from top left and down. Temperature readings were 20, 25, 27, 30 and 35 C.

19

Figure 15. Van’t Hoff plot separations of Flavanone enantiomers at 4 temperatures in 3 replicates.

The data collected had much less correlation than 3HP-1EE, which could be do to

changes in the column because it had just been hooked up to the pumps and detector.

Additional problems cold be due to the fact so of the readings were done on different

days. To test this readings would be taken at 7 different temperatures in duplicates of 2.

t t1 t2 w1 w2 k'1 k'2 R N T3.325 7.861 8.756 0.33 0.35 1.114 1.364 1.633 2.650 9147 213.329 7.813 8.692 0.30 0.33 1.113 1.347 1.611 2.819 11308 233.329 7.700 8.546 0.28 0.29 1.110 1.313 1.567 2.993 12557 253.329 7.642 8.479 0.30 0.31 1.110 1.296 1.547 2.767 10746 273.325 7.504 8.313 0.27 0.30 1.108 1.257 1.500 2.863 12359 303.333 7.438 8.224 0.27 0.27 1.106 1.231 1.467 2.943 12616 323.338 7.325 8.096 0.26 0.27 1.105 1.195 1.426 2.965 13215 35

Table 2. Data for Flavanone collected at 7 temperatures at 95 % hexane and 5% isopropanol.

20

Figure 16. Van’t Hoff Plot of Flavanone at 7 temperatures to determine H and S values using the ln vs 1/T (1/K).

The correlation is much better for there only being 2 duplicates at each

temperature. The slope of the line and y intercept is than used to fins the H and S

values. Using equation 19, the slope of 51.723 is used to determine the H, which is -

430 J. With equation 20, the y-intercept is used to determine the S value, which is -

0.570 J/K.

Conclusions:

Thermodynamic Separations of Chiral compounds using Normal Phase HPLC

gave interesting results. The two compounds tested, 3HP-1EE and Flavanone, are both of

21

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

different sizes. So when they were separated at a variety of temperatures different H

and S values should be obtained. As temperature increased for separation of 3HP-1EE

the retention time, capacity factor, resolution and selectivity factor decreased and the

number of theoretical plates remained constant. The values obtained showed that 3HP-

1EE is Enthalpy driven because of its large negative H and S values. It showed

that entropy is unfavorable, which is to be expected since it separated better at lower

temperatures.

As temperature for Flavanone enantiomeric separation increased the retention

time, capacity factor and selectivity factor decreased while the resolution and theoretical

plates increased. Flavanone had much smaller H and S values and may possibly be

entropy driven. This may be partially due to the size, creating steric effects in the

column. It was separated better at a high temperature suggesting that is not primarily

enthalpy driven.

The result of this shows that larger compounds are entropy driven and are more

easily separated at higher temperatures while smaller molecules are separated better at

lower temperatures because they are enthalpy driven. This result is specifically for a

polysaccharide column because inclusion complexes in amylose or cellulose chains. The

cavities created will have an affect on the larger molecules. As temperature of the

column is changed the conformation of the polysaccharide CSP will change in its

separation ability of the compound. These results are similar to Weng et al. where Amino

Acids and Binapthyl Compounds were examined.

22

References:

[1] Thompson, R. “A Practical Guide to HPLC Enantioseperations for Pharmaceutical

Compounds”, J. Liquid Chromatogr. & Related Technol., 2005, 28, 1215-1231.

[2] Wade, L.G. Organic Chemistry, Sixth Edition. Whitman College. Prearson Prentice

Hall: Chicago, 2006.

[3] 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, 2000, 52, 190-194.

[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.

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

[6] Yueqi, L; wenjian, L; Yuhua, Z; Shengxiang, J; Liren, C. Chromatographia. 52.

2000. 190-194.

[7] Macherey-Nagel, RESOLVOSIL BSA-7, Pretech Instruments, 2007,

<http://www.pretech.nu/products/HPLC/Spec_Resolvosil.htm>

[8] Jacobson, S.; Golshan-Shirazi, S.; Guiochon, G., Journal of Chromotography, 1990,

52, 23-36.

[9] Chiralpak IA. Chiral Technologies Product Literature, West Chester, PA. MORE

INFO

[10] Christian, Gary. Analytical Chemistry, 5th Edition, John Wiley and Sons, New York

1994, 505-544.

23

Appendix

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 Ch1-254nmflavano ne21C9-26-2008 1-19-06 PMflavano ne21C.dat

Retention TimeArea

Flavanone 21 C 95/5

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


Retention TimeArea

Flavanone 30 C 95/5

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


Retention TimeArea

Flavanone 35 C 95/5

24

Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

100

200

300

mAU

0

100

200

300

3.567

8202

3.725

1284

3.850

704

4.167

1299

4.308

2043

4.733

4294

5.017

2378

5.467

136

5.908

78467

6.400

387

6.708

2596913

7.100

26715518.008

17386

8.300

3410

8.733

11162

9.325

772


Retention TimeArea

Flavanone 21 C 90/10

Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

200

400

mAU

0

200

400

3.550

13931

3.817

3142

4.100

4166

4.233

5327

4.617

7763

4.825

5222

5.350

2059

5.758

90302

6.167

1974

6.500

2961448

6.817

30904587.975

24822

8.517

17173

8.950

4687

9.358

5849

9.533

2903


Retention TimeArea


Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

200

400

mAU

0

200

400

3.058

456

3.483

17631

3.725

5754

3.967

3833

4.100

5060

4.442

7190

4.758

3361

5.025

1349

5.542

103177

6.217

3279220

6.508

34483037.883

24528

8.183

14216

8.467

2005

9.517

1011


Retention TimeArea


Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

200

400

mAU

0

200

400

3.083

694

3.500

13479

3.733

3123

3.950

3131

4.083

5083

4.392

6891

4.717

4480

4.958

1015

5.142

529

5.483

80684

5.658

16699

6.108

3404116

6.383

3575162 7.433

4390

7.850

19170

7.992

16114

9.150

2386

SPD-20AV Ch1-254nmflavano ne35C10-13-2008 3-58-14 PMflavan on e35C.dat

Retention TimeArea


25

Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

200

400

mAU

0

200

400

3.383

6493

3.658

19757

3.850

7458

4.175

13547

4.492

16534

4.775

4787

4.942

14679

5.625

101453

6.192

3911370

6.417

42569717.600

10014

7.958

5293

8.333

4141

8.567

3109

9.183

2144


Retention TimeArea


Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

100

200

300

mAU

0

100

200

300

3.283

2454

3.608

13651

3.783

4426

4.083

7876

4.375

8127

4.675

2434

4.892

7337

5.483

56550

6.008

2351105

6.217

25536096.925

1823


Retention TimeArea


Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

100

200

300

mAU

0

100

200

300

3.283

2454

3.608

13651

3.783

4426

4.083

7876

4.375

8127

4.675

2434

4.892

7337

5.483

56550

6.008

2351105

6.217

25536096.925

1823


Retention TimeArea


Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

200

400

mAU

0

200

400

0.558

209

2.325

788

3.242

2190

3.542

15414

3.692

3524

3.800

2587

3.942

6124

4.175

9114

4.483

2453

4.800

6011

5.225

65901

5.658

2763183

5.842

3011779


Retention TimeArea


26

Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

20

40

60

mAU

0

20

40

60

3.100

50565

3.808

10858

3.942

16034

4.325

12206

4.633

11393

4.808

36783

5.375

638006

5.792

7073876.567

18697

SPD-20AV Ch1-254nmso ulsb y20C10-31-2008 2-03-55 PMsoulsby20C.d at

Retention TimeArea

3HP-1EE 21 C 85/15

Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

20

40

60

mAU

0

20

40

60

3.092

47145

3.775

14581

3.883

16650

4.550

34467

4.783

29869

5.275

631035

5.642

656247 6.092

59483

6.467

55010

7.150

57957

7.558

23249


Retention TimeArea

3HP-1EE 25 C 85/15

Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

25

50

75

mAU

0

25

50

75

3.067

53256

3.742

34182

4.450

39898

4.733

38484

5.150

670882

5.475

744556 6.075

48726

6.308

71298

6.892

31572

7.150

17893


Retention TimeArea

3HP-1EE 30 C 85/15

Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

20

40

mAU

0

20

40

3.050

31247

3.308

3766

3.700

22779

4.083

6891

4.367

19704

4.717

27960

5.067

395315

5.350

4234805.708

27140

6.050

27311

6.167

54725

6.883

22569


Retention TimeArea

3HP-1EE 35 C 85/15

27

Minutes

0 1 2 3 4 5 6 7 8 9 10

mAU

0

20

40

60

mAU

0

20

40

60

3.150

58175

3.742

4479

3.908

3298

4.108

8357

4.892

6191

5.058

1455

5.242

470

5.367

326

5.675

5262

6.575

626419

7.217

622909

7.967

4859

SPD-20AV Ch1-254nmso ulsb y20C11-3-2008 1-49-40 PMsou lsb y20C.dat

Retention TimeArea

3HP-1EE 21 C 90/10

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


Retention TimeArea

3HP-1EE 25 C 90/10

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


Retention TimeArea

3HP-1EE 30 C 90/10

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


Retention TimeArea

3HP-1EE 35 C 90/10

28

Table A1. Experimentally determined values of 3HP-1EE.3HP-1EE 95/5

20 C 25 C 30 C 35 Ca k'k' RN

3HP-1EE 90/10a 1.100 1.089 1.081 1.073k' 1.023 1.060 1.009 0.956k' 1.225 1.244 1.172 1.052R 1.804 1.717 1.566 1.280N 6351.61 9855.80 6266.70 4993.24

3HP-1EE 85/15a 1.077 1.071 1.063 1.057k' 0.597 0.709 0.679 0.655k' 0.668 0.830 0.785 0.548R 1.105 0.763 1.168 1.104N 2665.58 2053.83 7367.36 5708.64

Table A2. 3HP-1EE values for calibration curve.3HP-1EE 95/5

293.15 K 298.15 K 303.15 K 308.15 Kln a1/T 0.0034 0.0034 0.0033 0.0032

3HP-1EE 90/10ln a 0.096 0.085 0.078 0.0701/T 0.0034 0.0034 0.0033 0.0032

3HP-1EE 85/5ln a 0.075 0.068 0.061 0.0551/T 0.0034 0.0034 0.0033 0.0032

Table A3. Determined H and S values. slope y-int delta-delta H delta-delta S95/5

90/10 149.64 0.4156 -1244.18178 3.4555062

85/15 116.85 0.324 -971.549325 2.693898

29

Figure A1. Calibration Curve for 3HP-1EE with 90/10 hexane to isopropanol.

3HP-1EE 90/10

y = 149.64x - 0.4156R2 = 0.9949

0.000

0.020

0.040

0.060

0.080

0.100

0.120

0.0032 0.0033 0.0033 0.0034 0.0034 0.00351/T

ln a

Figure A2. Calibration Curve for 3HP-1EE with 55/15 hexane to isopropanol.

3HP-1EE 85/15

y = 116.85x - 0.324

R2 = 0.9992

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.0032 0.0033 0.0033 0.0034 0.0034 0.00351/T

ln a

30

Table A4. Experimentally determined values for Flavanone.Flavanone 95/5

20 C 25 C 30 C 35 Ca 1.105 1.092 1.091k' 1.264 1.235 1.183k' 1.499 1.438 1.364R 2.607 2.400 2.500N 11672.93964 12988.98556 13333.78278

Flavanone 90/10a 1.064 1.049 1.047 1.045k' 0.888 0.831 0.792 0.739k' 1.010 0.921 0.877 0.817R 2.487 1.306 1.274 0.974N 11491.84 12614.22 13049.95 13464.84

Flavanone 85/15a 1.036 1.035 1.034 1.033k' 0.696 0.666 0.631 0.603k' 0.758 0.724 0.692 0.656R 0.842 0.971 0.924 0.922N 8488.55 12268.58 13768.68 14188.57

Table A5. Flavanone values for calibration curve.Flavanone 95/5

293.15 K 298.15 K 303.15 K 308.15 Kln a 0.100 0.088 0.0871/T 0.0034 0.0034 0.0033 0.0032

Flavanone 90/10ln a 0.062 0.048 0.046 0.0441/T 0.0034 0.0034 0.0033 0.0032

Flavanone 85/5ln a 0.035 0.034 0.033 0.0321/T 0.0034 0.0034 0.0033 0.0032

Table A6. Determined H and S values slope y-int delta-delta H delta-delta S95/5 83.11 0.184 -691.018095 1.529868

90/10 101.83 0.2889 -846.665535 2.40205905

85/15 17.622 0.0249 146.518119 0.20703105

31

Figure A3. Calibration Curve for Flavanone with 95/5 hexane to isopropranol.

Flavanone 95/5

y = 83.11x - 0.184R2 = 0.9347

0.0840.0860.0880.0900.0920.0940.0960.0980.1000.102

0.0032 0.0033 0.0033 0.0034 0.0034 0.00351/T

ln a


Flavanone 90/10

y = 101.83x - 0.2889R2 = 0.7869

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.0032 0.0033 0.0033 0.0034 0.0034 0.00351/T

ln a

32


Flavanone 85/15

y = 17.622x - 0.0249R2 = 0.9737

0.032

0.033

0.033

0.034

0.034

0.035

0.035

0.036

0.036

0.0032 0.0033 0.0033 0.0034 0.0034 0.00351/T

ln a

33

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Senior Thesis FINAL 1