Fluoro-methylphenylcarbamates of Cellulose as

Chiral Stationary Phases for Supercritical Fluid

Chromatography

William Farrell1 and Matthew Przybyciel2

1Pfizer Global R&D - La Jolla, CA 2ES Industries - West Berlin, NJ

ABSTRACT

The use of aromatic halogenated substituents within carbohydrate

chiral stationary phases is not a new concept, with early work

dating back to the mid to late 1990’s1,2. Chloro-, bromo-, and

fluoro-methylphenylcarbamates all showed chiral recognition, and

there are several chloro-phenyl substituted versions available

commercially. However, with the resurgence of fluorinated

chemistry being used for drug development, the need for phases

suitable for chiral recognition of fluorinated compounds is

facilitated by re-introduction of these types of halogenated

phases. Introduction of a fluorophilic retention mechanism can be

particularly useful in support of medicinal chemistry drug

discovery efforts, where more than a third of newly approved

small molecule drugs contain fluorine3,4.

Two new fluorinated phases, 4-fluoro-3-methylphenylcarbamate

and 2-fluoro-5-methylphenylcarbamate, were prepared on

cellulose and evaluated for use with drug-like compounds

containing fluorine constituents. Separations of a wide variety of

these compounds will be presented as well as comparative

separations using other halogenated stationary phases.

Introduction

Traditional Phases

Chiral SFC is the primary tool for separation of enantiomers for

medicinal chemistry support. Among the chiral selectors are the

popular polysaccharides phases, which are substituted phenyl

moieties bonded to either amylose or cellulose.

Figure 6: Analytical chromatograms of the separation of

MFCD26517069 using HPLC (top) and SFC (bottom)

conditions.

A non-ideal separation was achieved using HPLC conditions

and a traditional phase (top), but was extremely sensitive to

temperature and thus difficult to scale for purification.

Using a fluorinated phase, complete separation was

achieved using SFC conditions. Significant savings in solvent

and time were achieved upon scale-up to purification. Only a

small percentage of methanol was added post-column to

facilitate collection.

Figure 8: Effect of dipole of moment of stationary phase on

the separation of MFCD265170695,6

Selectivity (α) and resolution (R) values for each separation

are included. Dipole moment values provided reflect the

magnitude differences in an ideal system and were not

measured for each specific phase. a) Cellulose tris-(4-chloro-

3-methylphenylcarbamate) phase (CC4). b) Cellulose tris-(4-

fluoro-3-(trifluoromethyl)phenylcarbamate (CCO-F4-CF3)

phase. c). Cellulose tris-(4-fluoro-3-methylphenylcarbamate)

phase (CCO-F4).

Figure 1: Common phases used for chiral separations

Typical mobile phases used are limited to alcohols combined

with hydrocarbons (e.g. hexanes or heptane in HPLC) or carbon

dioxide (SFC).

Additional phases commonly used include Pirkle-type (such as

the Whelk-O1) and peptide macrocyclic phases. These have a

broader solvent compatibility range, but are usually applied in

reverse phase mode (aqueous/organic) or in polar organic mode

(e.g. 20mM ammonium acetate in methanol), as well as in SFC.

REFERENCES

1. Enantioseparation on fluoro-methylphenylcarbamates of cellulose and amylose as chiral

stationary phases for high performance liquid chromatography, Yashima et.al, Polymer

Journal, Vol 27, No. 8, pp 856-861 (1995).

2. 3-Fluoro-, 3-chloro- and 3-bromo-5-methylphenyl carbamates of cellulose and amylose as

chiral stationary phases for high performance liquid chromatographic enantioseparation, B.

Chankvetadze, et al, J. Chrom. A, 787, (1997) 67-77.

3. Fluorous Reverse Phase Silia Ge: A New Tool for Preparative Separations in Synthetic Organic

and Organofluorine Chemistry, Curran, D. P., Synlett 2001, (9), 1488-1496.

http://dx.doi.org/10.1055/s-2001-16800

4. A Bountiful Year, Jarvis, L. M., Chemical & Engineering News 2013, 91, (5), 15-17

http://dx.doi.org/10.1021/cen-09105-bus1

5. Each column is 4.6mm I.D. x 250mm length containing 5-µm particles maintained at 25°C. The

mobile phase consists of CO2 and percentage co-solvent listed in each chromatogram

delivered at a flow rate of 3.0mL/min with 160 bar outlet pressure.

6. Alkyl Halides. Retrieved from http://crab.rutgers.edu/~alroche/Ch06.pdf

CONCLUSION

The use of fluorine for chiral stationary phases has demonstrated

utility and appears comparable to commercial chlorinated phases.

The magnitude of the selectivity appears to derive from changes in

the dipole of the benzylic functional groups.

Nonetheless, the fluorinated versions offer several advantages in

potential savings in solvent consumption and reduced retention

time.

The introduction of the CF3 functionality opens a door for further

exploration of commercial phases by substituting the methyl group.

Liquid CO2 can be used to enhance selectivity.

Figure 9: The overall trend in selectivity (α) and resolution (R)

for pharmaceutical compounds supports effectiveness of dipole

moment manipulation on the stationary phase5

As shown in Figures 8 & 9, the addition of electron-withdrawing

functional groups plays a role in the separation mechanism, with

the greatest overall effect achieved with chlorine.

Cellulose tris-(4-chloro-3-methylphenylcarbamate) = 4Cl-3M

Cellulose tris-(4-fluoro-3-(trifluoromethyl)phenylcarbamate = 4F-CF3

Cellulose tris-(4-fluoro-3-methylphenylcarbamate) = 4F-3M

Figure 10: Example chromatograms of metoprolol

enantiomers on each of the commercial fluoro-methyl and

chloro-methyl phases5

Further demonstrating the effect of the phase dipole, selectivity

of metoprolol enantiomers can be enhanced by substituting the

halogen (CCO-F4 to CC4), the methyl group (CCO-F4 to CCO-

F4-CF3), or the relative position of both the halogen and methyl

groups (CCO-F4 to CCO-F2).

18 minutes2 4 6 8 10 12 14 16

mAU

0

10

20

30

40

50

60

70

4.5

01

7.377

14.74015.796

4.5 minutes0.5 1 1.5 2 2.5 3 3.5 4

mAU

0

50

100

150

200

250

300

350

3.099

3.355

Column: Chiralcell OJ-H

Column: 4.6x250mm, 5µm

Mobile Phase: 1% IPA in Heptane

Flow rate: 1.0mL/min

Temp: 0°C

Inj vol: 3µL

Column: CCO-F4

Column: 4.6x250mm, 5µm

Mobile Phase: 100% CO2

Flow rate: 3.0mL/min

Pressure: 200bar

Temp: 10°C

Inj vol: 3µL

O

OR

O*

RORO

*n

R =

O

Commercial OJ-H Cellulose tris(methyl benzoate)

Silica gel

O

OR

O*

RORO

*n

R =

O

N F

Silica gel

Custom Cellulose tris(4-fluoro-3-

methylphenylcarbamate)

O

O

F F

X = C-Cl µ= 1.56D

X = C-F µ= 1.51D

α = 1.19R = 3.97

O

O

F F

5.185

6.182

4.129

4.649

min1 2 3 4 5 6 7

3.0763.320

α = 1.12R = 2.89

α = 1.08R = 2.53

CC44Cl-3M

CCO-F4-CF34F-3CF3

CCO-F44F-3M

MFCD26517069

a)

b)

c)

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

R

4F-3M 4F-3CF3 4Cl-3M

1.05

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

alp

ha

Overall trend for selectivity and resolution

4F-3M < 4F-CF3 < 4Cl-3M

Selectivity

Resolution

NH

ONH

OH

CH3

CH3

Pindolol

NH

ONH

OH

CH3

CH3

Propranolol

O O

OH O

CH3

Warfarin

O NH

CH2

OH

CH3

CH3

Alprenolol

NHO

OH

OCH3 CH3

CH3

Metoprolol

N

N

O Cl

Cl

Cl

Cl

N

O

O

O

O

CH3

CH3

CH3

CH3

CH3

CH3

N

CH3

Miconazole

Verapamil

NH2

OONHCH3

CH3

OH

Atenolol

5.387

8.167

4.223

5.757

min2 4 6 8 10 12 14

3.282

4.230

3.948

4.332

CCO-F4-CF3

CCO-F4

CCO-F2

ES CC4

O

OR

O*

RORO

*n

R =

O

N F

Silica gel

Cellulose tris(4-fluoro-3-methylphenylcarbamate)

R =

O

N

F

Cellulose tris(2-fluoro-5-methylphenylcarbamate)

Warfarin20% MeOH

min0.5 1 1.5 2 2.5 3 3.5

1.887

2.395

min0 0.5 1 1.5 2 2.5 3

2.260

2.523PF Compound20% MeOH

Miconazole25% 20mM AMF in MeOH

min0.5 1 1.5 2 2.5

1.799

1.955

min0.5 1 1.5 2 2.5

1.976

2.155Verapamil25% 20mM AMF in MeOH

O O

OH O

CH3

N

O

O

O

O

CH3

CH3

CH3

CH3

CH3

CH3

N

CH3

N

N

O Cl

Cl

Cl

Cl

Miconazole25% 20mM AMF in MeOH

Verapamil25% 20mM AMF in MeOH

N

O

O

O

O

CH3

CH3

CH3

CH3

CH3

CH3

N

CH3

N

N

O Cl

Cl

Cl

Cl

O

OR

O*

RORO*

n

R =

F

CF3

O

Silica gel

Cellulose tris(4-fluoro-3-trifluoromethylphenylbenzoate)

R =

F

F

O

Cellulose tris(3,4-Difluoro-methylphenylbenzoate)

1.366 1.409

1.303

min0.5 1 1.5 2 2.5

1.364

Inj # 1

Inj # 3

Inj # 5

trans stilbene

oxide

O

min0 0.5 1 1.5 2 2.5 3 3.5

2.557

2.808

min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

3.029

3.499

Figure 3: Example separations using 1st Generation

fluorinated phases5

Expecting to capitalize on fluorophillic interactions, two

prototype phases were made which demonstrate versatility

against a broad selection of compounds.

Figure 7: Example separations on the 2nd Generation

prototype fluorinated phases5

Following on the success of the CCO-F4 phase, two

additional phases were designed to probe different dipole

interactions, of which only one demonstrated versatility

against a broad selection of compounds. The difluorinated

column showed significant bleed and loss of selectivity even

with low percentages of solvent (<5%).

Chiralpak+ AD

Coated -

Amylose

Chiralpak AS

Coated -

Amylose

Chiralpak IC

Immobilized -

Cellulose

Chiralcel OJ

Coated -

Cellulose

Chiralcel OD

Coated -

Cellulose

Lux++ Cellulose-2

Coated -

Cellulose

Lux Cellulose-4

Coated -

Cellulose

Lux Amylose-2

Coated -

Amylose

Whelk-O1Bonded

Pirkle type

ES IndustriesCC4*

Coated -

Cellulose

N

O

R

CH 3

CH 3

CH3

N

O

R

N

O

R

Cl

Cl

O

R

N

O

R

CH 3

CH 3

N

O

R

Cl

CH3

N

O

R

CH3

Cl

N

O

R

Cl

C H3

N

O

R

CH 3

Cl

Si

CH 3CH 3

N

ONO 2

NO 2

O

Column Column type Chiral SelectorColumn Column type Chiral Selector

Figure 2: Chiral phases used in purification support of

medicinal chemistry

As shown in Figure 2, an increasing number of chiral

compounds achieve separation on any of these traditional

phases, especially those compounds with low molecular weight

(i.e. MW < 200 Da) or have limited complexity/functionality near

the chiral center. Gas chromatography is probably best suited for

these compounds; however, it is inconsistent with the goals of

isolating pure enantiomers at any workable scale for medicinal

chemistry support. Ideally, successful separations of the

enantiomers would have a selectivity of >1.1 and a minimum

resolution >1.5.

Figure 4: Example chromatograms of MFCD26517069 using

standard SFC conditions with 1 % acetonitrile as modifier.

Inset: Yellow region represents normal operating conditions

in SFC. Blue region represents rarely used region in SFC.

Using MFCD26517069 as a probe compound, no separation

of enantiomers was initially obtained using any traditional

phases in SFC mode. In addition, the prototype fluorinated

phases also appeared to be ineffective.

Since the compound is very non-polar, mobile phases of

hydrocarbons (HPLC) and carbon dioxide (SFC) were also

explored.

Figure 5: Chromatograms of MFCD26517069 at various

pressure and temperature settings

Borrowing from some the partial success using HPLC (see

Figure 6), modification of the density of the mobile phase in

SFC mode did result in enantiomeric separation. Figure 5

shows the effects of temperature and pressure on the

separation as the conditions move from the yellow to blue

regions indicated in the Figure 4 inset.

25°C

Inlet: 315bar

Outlet: 150bar

Inlet: 370bar

Outlet: 200bar

Inlet: 420bar

Outlet: 250bar

Inlet: 462bar

Outlet: 300bar

Inlet: 505bar

Outlet: 350bar

min0.5 1 1.5 2 2.5 3 3.5

10°C

Inlet: 318bar

Outlet: 150bar

Inlet: 370bar

Outlet: 200bar

Inlet: 420bar

Outlet: 250bar

Inlet: 464bar

Outlet: 300bar

Inlet: 510bar

Outlet: 350bar

Inlet: 550bar

Outlet: 400bar

min0.5 1 1.5 2 2.5 3 3.5

283 298

480

150

323

350

Bar

Kelvin

Celsius

Lines Reflect Density

Region A = Liquid

Yellow Region = “Sweet Spot”

Region C = Low Density

25 5010

PSI

7000

5000

2000

Region B = High Density

Blue Region = Interesting…

min1 2 3 4

mAU

0

20

40

60

80

100

120 1.732

min1 2 3 4

mAU

0

25

50

75

100

125

150

175

200 1.868

O

O

F F

Columns: 4.6x250mm, 5µm

Modifier: 1% MeCN

Flow rate: 3.0mL/min

Pressure: 160bar

Temp: 25°C

Inj vol: 3µL

CCO-F4

CC4

MFCD26517069

Prototype Phases – 1st Generation

Prototype Phases – 2nd Generation

Metoprolol25% 20mM Ammonia in IPA

CCO-F4 CCO-F2

CCO-F4-CF3 CCO-DiF

CCO-F4