Column Wa t c h C o l u m n - Pharmaceutical Technology

1 6 LCGC NORTH AMERICA VOLUME 23 NUMBER 1 JA N UA RY 2005 w w w. c h r o m a t o g r a p h y o n l i n e . c o m 

C Column o l u m n 

Wa t c h 

P reparative Chiral SFC as a 

G reen Technology for Rapid 

Access to Enantiopurity in 

Pharmaceutical Process Researc h 

Guest Authors 

Christopher J. We l c h * , 

William R. Leonard Jr. , 

Jimmy O. DaSilva, 

Mirlinda Biba, Jennifer 

A l b a n e z e - Wa l k e r, Dere k 

W. Henderson, Brian 

Laing, and David J. 

M a t h re 

This month’s installment 

of “Column Wa t c h ” 

p rovides an overview of 

g reen chemistry 

considerations for the 

k i l o g r a m - s c a l e 

p re p a r a t i v e 

c h ro m a t o g r a p h i c 

separation of 

enantiomers, focusing 

especially on re c e n t 

results from the authors’ 

laboratories, illustrating 

solvent and time savings 

realized with pre p a r a t i v e 

s u p e rcritical fluid 

c h romatography (SFC). 

They compare optimized 

high performance liquid 

c h romatography and SFC 

in terms of energy 

e fficiency and 

t h ro u g h p u t . 

Ronald E. Majors 

Column Watch Editor 

Re c e n t l y, the subject of “g re e n 

c h e m i s t ry” has re c e i ved considerable 

attention from academic and 

industrial re s e a rchers alike (1). While the 

central tenets of waste reduction, pro c e s s 

e c o n o m y, and elimination of risks and haza 

rds have long been embraced by industrial 

p rocess chemists, the current focus on gre e n 

c h e m i s t ry concerns has led to re n ewe d 

a p p reciation for the importance of these 

topics. The analysis of the “g re e n n e s s” of 

any given process, operation, or methodology 

is a useful exe rcise that can lead to 

p rocess improvement and refinements. T h i s 

e xe rcise can be particularly valuable for new 

technologies that might not yet have a long 

track re c o rd of practical utilization. Pre p a r- 

a t i ve chromatographic resolution of enantiomers 

is one such emerging technology 

that recently has become widely used for 

p roviding rapid access to enantiopure materials 

to support pharmaceutical deve l o p- 

ment. He re we provide an ove rv i ew of 

g reen chemistry issues relating to pre p a r a- 

t i ve chro m a t o g r a p h y, concentrating especially 

on the green advantages of pre p a r a- 

t i ve supercritical fluid chro m a t o g r a p h y 

(SFC), in which carbon dioxide re p l a c e s 

the flammable and toxic petro c h e m i c a l - 

d e r i ved hyd rocarbon solvents typically used 

in pre p a r a t i ve liquid chro m a t o g r a p h y. 

Can Preparative Chro m a t o g r a p h y 

Be Gre e n ? 

First impressions might suggest that pre p a r- 

a t i ve chro m a t o g r a p h y, with its intensive use 

of solvent, is decidedly “u n - g reen.” Howe 

ve r, economic factors usually dictate that 

industrial-scale chromatographic pro c e s s e s 

e m p l oy solvent re c ycling (2). In addition, 

the smaller scale pre p a r a t i ve chro m a t o g r a- 

phy performed in support of deve l o p m e n t a l 

re s e a rch can sometimes eliminate the need 

for developing and carrying out more traditional 

chemical syntheses, there by saving 

considerable labor and time (3), and sometimes 

even resulting in a net decrease in 

waste generation. Thus, the use of pre p a r a- 

t i ve chromatography can sometimes be a 

“g re e n e r” approach to conventional deve l- 

opment. Fu rt h e r m o re, newer forms of 

p re p a r a t i ve chro m a t o g r a p h y, such as SFC, 

can be viewed as an even greener alternative 

to classical pre p a r a t i ve chro m a t o g r a p h y. 

( Please see the sidebar on the follow i n g 

p a g e . ) 

Chirality and the 

Pharmaceutical Industry 

Most pharmaceuticals are chiral; that is, 

they can exist as either of two nonsuperimposable 

mirror image forms, termed enantiomers. 

Although the importance of chirality 

has been appreciated and addressed by 

the pharmaceutical industry for decades, it 

is only within the past few years that a shift 

t ow a rd development of most chiral pharmaceutical 

candidates as single enantiomers 

has occurred. As technologies for measuring 

and making enantiopure materials have 

i m p roved, the production of enantiopure 

pharmaceuticals has become commonplace, 

with many of the top-selling drugs in the 

world now being sold in enantiopure form. 

C o n s e q u e n t l y, the subject of chirality and 

the pharmaceutical industry is a topic of 

considerable recent interest and import a n c e 

( 4 – 6 ) . 

P reparative Chromatography in 

Organic Synthesis: Realizing the 

Wo o d w a rd Vi s i o n 

Pre p a r a t i ve chiral chromatography is being 

used increasingly in pharmaceutical deve l-

1 8 LCGC NORTH AMERICA VOLUME 23 NUMBER 1 JA N UA RY 2005 

12 Principles of 

G reen Chemistry 

(From reference 1) 

1.Prevention: It is better to prevent waste 

than to treat or clean up waste after it has 

been created. 

2. Atom Economy: Synthetic methods 

should be designed to maximize the incorporation 

of all materials used in the 

process into the final product. 

3. Less Hazardous Chemical Syntheses: 

Wherever practicable, synthetic methods 

should be designed to use and generate 

substances that possess little or no toxicity 

to human health and the environment. 

4. Designing Safer Chemicals: Chemical 

products should be designed to eff e c t 

their desired function while minimizing 

their toxicity. 

5. Safer Solvents and Auxiliaries: The use 

of auxiliary substances (for example, solvents, 

separation agents, and so forth) 

should be made unnecessary wherever 

possible and innocuous when used. 

6. Design for Energy Eff i c i e n c y : E n e r g y 

requirements of chemical processes should 

be recognized for their environmental and 

economic impacts and should be minimized. 

If possible, synthetic methods 

should be conducted at ambient temperature 

and pressure. 

7. Use of Renewable Feedstocks: A raw 

material or feedstock should be renewable 

rather than depleting whenever technically 

and economically practicable. 

8. Reduce Derivatives: Unnecessary derivatization 

(use of blocking groups, protection–deprotection, 

temporary modification 

of physical–chemical processes) 

should be minimized or avoided if possible, 

because such steps require additional 

reagents and can generate waste. 

9. Catalysis: Catalytic reagents (as selective 

as possible) are superior to stoichiometric 

r e a g e n t s . 

10. Design for Degradation: Chemical 

products should be designed so that at 

the end of their function they break down 

into innocuous degradation products and 

do not persist in the environment. 

11. Real-time Analysis for Pollution Prev 

e n t i o n : Analytical methodologies need to 

be further developed to allow for realtime, 

in-process monitoring and control 

before the formation of hazardous subs 

t a n c e s . 

12. Inherently Safer Chemistry for Accident 

Prevention: Substances and the form 

of a substance used in a chemical process 

should be chosen to minimize the potential 

for chemical accidents. 

opment for rapidly accessing enantiomerically 

pure materials on the kilogram or eve n 

larger scale (7). Pre p a r a t i ve high perf o r m- 

ance liquid chromatography (HPLC) is 

used most frequently for the kilogram-scale 

separations needed to support deve l o p- 

ment, with simulated moving bed chromatography 

(SMB) or other multicolumn 

c h romatography approaches pre d o m i n a t i n g 

at industrial scale (greater than hundreds of 

kilograms) (8–12).The integration of 

p re p a r a t i ve chiral chromatography into 

organic synthesis remains a rapidly evo l v i n g 

field, and new stratagems for merging the 

two fields are being created. Ne ve rt h e l e s s , 

the incorporation of pre p a r a t i ve HPLC 

into organic synthesis is by no means a new 

phenomenon. R.B. Wo o d w a rd, perhaps the 

most famous synthetic chemist of the twe n- 

tieth century, was an early advocate of 

p re p a r a t i ve HPLC, declaring in 1973 (2) 

that “The power of these high pre s s u re liquid 

chromatographic methods hardly can 

be imagined by the chemist who has not 

had experience with them; they re p re s e n t 

re l a t i vely simple instrumentation and I am 

c e rtain that they will be indispensable in 

the laboratory of eve ry organic chemist in 

the near future.” (13) 

In t e re s t i n g l y, it is only within the last few 

years that Wo o d w a rd’s prediction of the 

w i d e s p read adoption of pre p a r a t i ve HPLC 

as an enabling technique for organic synthesis 

has begun to be borne out. In re c e n t 

years, there has been a growing appre c i a t i o n 

w w w. c h r o m a t o g r a p h y o n l i n e . c o m 

F i g u re 1: A typical case illustrating the preparative chromatographic enantioseparation of a 

development intermediate on the kilogram scale. Column: 25 cm 3 11 cm DAC Chiralpak AD; flow 

rate: 600 mL/min; mobile phase: 25% ethanol–heptane; detection: UV absorbance at 285 nm; injection: 

150 mL racemate at 83 mg/mL in mobile phase. 

of the value that pre p a r a t i ve chro m a t o g r a- 

phy can bring to organic synthesis, and the 

technique is now used broadly within the 

pharmaceutical and fine chemical 

industries. 

G reen Aspects of Pre p a r a t i v e 

C h romatography: Waste Reduction 

Se veral of the 12 principles of green chemi 

s t ry, outlined in the sidebar, are especially 

applicable to the field of pre p a r a t i ve chrom 

a t o g r a p h y. Process chemists have long 

been concerned with the topics of waste 

p re vention, atom economy, and the use of 

i n h e rently safe chemistry for accident prevention 

(principles 1, 2, and 12). Also, 

p re p a r a t i ve chromatography is concerned 

especially with the issue of safer solve n t s 

and auxiliaries (principle 5). 

Elimination of waste is always a key 

g reen chemistry concern, and an import a n t 

factor in any separation of enantiomers 

(that is, resolution). Whether using classical 

resolution via diastereomeric salt formation, 

enzymatic kinetic resolution, pre f e re n t i a l 

c rystallization, or chro m a t o g r a p h y, all re s o- 

lutions suffer, in theory, from the fundamental 

drawback of being inherently wasteful, 

as at most, only half of the material is 

re c ove red (the half corresponding the 

d e s i red enantiomer, the other half being 

“w a s t e”). Despite this seemingly gross violation 

of the principles of atom economy 

(14), racemization and re c ycling of the 

u n d e s i red enantiomer sometimes is possi-


F i g u re 2: Representative chromatograms illustrating (a) a single injection and (b) repetitive overlapped injections for the semipreparative SFC resolution 

of the enantiomers of a developmental compound. Column: 25 cm 3 2 cm Chiralcel OD; mobile phase: 32% (25 mM isobutylamine in 

methanol)–carbon dioxide; flow rate: 50 mL/min; outlet pressure: 100 bar; detection: UV absorbance at 280 nm; injection: 2 mL at 160 mg/mL. 

ble, enabling higher yield and reduction of 

waste. Coupling of such resolution and isomerization 

approaches can lead to tru l y 

i m p re s s i ve processes for generating enantiopurity 

(15), and such approaches have 

long been a mainstay of successful industrial-scale 

synthesis of enantiopure materials. 

The possibility of racemizing and re c y- 

cling the undesired enantiomer from a 

c h romatographic resolution is becoming a 

routine consideration in development, and 

when possible, such racemization–re c yc l i n g 

a p p roaches can have a significant impact on 

p rocess economy. T h e re f o re, inve s t i g a t i o n 

of racemization is an important consideration 

when deciding where in a synthesis a 

resolution should be placed. 


C h romatography: Safer Solvents 

The green chemistry principle of using safer 

s o l vents and auxiliaries is critically important 

to the area of pre p a r a t i ve chro m a t o g r a- 

p h y. A typical pre p a r a t i ve HPLC re s o l u t i o n 

is illustrated in Fi g u re 1. In this separation, 

the desired component is the second eluted 

e n a n t i o m e r, which is collected with . 9 8 % 

enantiomeric excess and 80% re c ove ry. 

Re p e t i t i ve injection under these conditions 

was used to obtain 1.1 kg of desired enantiomer 

with 55 h of instrument time and 

with the utilization of about 2000 L of solvent, 

840 L of which was evaporated for an 

overall productivity of about 0.3 kkd (kilograms 

of purified enantiomer per kilogram 

of stationary phase per 24-h day). Although 

the conditions of this pre p a r a t i ve separation 

could be improved to afford improved productivity 

and re c ove ry, it is important to 

re a l i ze that accessing more than a kilogram 

of enantiopure product in only 55 h means 

the chromatographic approach can be performed 

with only a small fraction of the 

cost that would be re q u i red to obtain this 

same result by conventional methods. It is 

this economic reality that underlies the 

w i d e s p read adoption of pre p a r a t i ve chromatography 

within the pharmaceutical and 

fine chemical industries. 

Productivity is the key metric for pre p a r- 

a t i ve chiral chromatography and is give n 

with units of kkd. In early deve l o p m e n t , 

c h romatographic productivity is often poor 

(0.1 kkd or even lower) with a good separation 

having a productivity in the range of 1 

kkd. A truly re m a rkable separation might 

h a ve a productivity greater than 10 kkd. 

From the example shown in Fi g u re 1, it can 

be appreciated that a large amount of solvent 

(2000 L) is re q u i red for perf o r m i n g 

this re l a t i vely unpro d u c t i ve separation, and 

while this solvent can in principle be re c y- 

cled, this typically is not done during early 

d e velopment re s e a rch. In addition to being 

s o m ewhat wasteful of re s o u rces, the use of 

so much solvent re q u i res specialized equipment 

and work environment, and dictates 

that a ve ry large volume of solvent must be 

e vaporated to re c over the desired material. 

The use of SFC for pre p a r a t i ve enantioseparation 

has enjoyed considerable 

recent attention (16–19) and is the method 

of first choice in our own laboratories. In 

this technique, supercritical or subcritical 

carbon dioxide replaces flammable and 

t oxic petro c h e m i c a l - d e r i ved hyd ro c a r b o n s , 

resulting in reduction in solvent utilization 

by as much as 90% or more. The re s u l t i n g 

d e c rease in solvent use and waste generation 

offers a green advantage with an economic 

bonus that makes pre p a r a t i ve SFC 

especially attractive. Fu rt h e r m o re, with 

p re p a r a t i ve SFC, the product is re c ove re d 

in a more concentrated form re l a t i ve to 

H P LC, greatly reducing the amount of solvent 

that must be evaporated and re s u l t i n g 

in considerable savings in labor, time, and 

energy costs. Fi n a l l y, because of the low viscosity 

of the supercritical fluid eluent, separations 

can be conducted at flow rates that 

would be impossible with liquid solve n t s , 

an advantage that can contribute to the 

often higher productivity of pre p a r a t i ve 

SFC enantioseparations re l a t i ve to HPLC 

methods. Cu m u l a t i ve l y, these adva n t a g e s 

make pre p a r a t i ve SFC enantioseparation an


F i g u re 3: Semipreparative SFC used for preparative separation of enantiomers. More than 200 g of a racemate was resolved in ;2700 injections, 

with ;60 h of instrument time. Chiralcel OF (Chiral Technologies) chromatograms showing (top) three repeat injection cycles and (bottom) a block 

of 99 injections. Productivity 5 0.8 kkd. Column: 25 cm 3 2 cm Chiralcel OF; 15% isopropanol–carbon dioxide; flow rate: 50 mL/min; outlet pressure: 

100 bar; detection: UV absorbance at 270 nm; injection: 500 mL at 150 mg/mL in isopropanol. 

a t t r a c t i ve and potentially greener addition 

to conventional HPLC approaches, and a 

technique with a promising future. 


C h romatography: Energy 

E ff i c i e n c y 

Energy efficiency is another matter of considerable 

importance in large-scale chromatographic 

separations. Pre p a r a t i ve liquid 

c h romatography using automated fraction 

collection and solvent re c ycling via continuous 

distillation was known by the early 

1970s (20) and is increasingly used today. 

So l vent evaporation can be highly energy 

i n t e n s i ve, to the point that the energy 

re q u i rements for evaporation can become 

the dominant cost in an industrial-scale 

c h romatographic process. An SFC system 

re q u i res additional heating and cooling not 

re q u i red for HPLC systems, and thus, additional 

energy demands. Incoming carbon 

d i oxide eluent must be cooled to a liquid so 

as to allow effective pumping, and immediately 

following pumping, the temperature 

of the carbon dioxide stream typically is 

raised to about 35 °C before it enters the 

column. Eva p o r a t i ve cooling resulting fro m 

d e p ressurization at the postcolumn outlet 

s t ream of the SFC instrument must be 

counteracted by more heating, which can 

be fairly energy intensive. Fi n a l l y, re c yc l i n g 

carbon dioxide re q u i res additional cooling 

of carbon dioxide gas from the outlet 

s t ream so as to condense into a liquid for 

reuse. In total, an operating SFC unit is 

heated and cooled simultaneously at seve r a l 

locations, and although in principle some 

o p p o rtunities exist for reduction of energy 

consumption via heat exchange, this typically 

is not done on laboratory-scale instruments. 

In comparison, HPLC re q u i res no 

heating and cooling for operation, raising 

the question of the energy utilization of 

SFC re l a t i ve to HPLC. The power demand 

is less than 5 kW for a pre p a r a t i ve SFC system 

pumping 350 g/min of a 10% 

methanol–carbon dioxide eluent mixture at 

a temperature of 35 °C and an outlet pre s- 

s u re of 100 bar with carbon dioxide re c y- 

cling. Power re q u i rements for continuous 

s o l vent evaporation (heating and cooling) 

to keep pace with such a unit would be on 

the order of 2 kW, whereas power re q u i rements 

to keep pace with solvent eva p o r a- 

tion for a comparable HPLC unit (assuming 

103 s o l vent utilization) would be on 

the order of 20 kW. Thus, it can be seen 

that even in terms of energy utilization, 

SFC is greener than HPLC, because of the 

high energy cost of solvent evaporation. As 

later examples will show, this SFC adva n- 

tage can be even more pronounced because 

of additional solvent reductions afforded by 

the oftentimes improved productivity of 

SFC versus HPLC. 


C h romatography: Te m p e r a t u re , 

P re s s u re, and Carbon Dioxide as a 

G reenhouse Gas 

In addition to the fundamental point of 

designing for energy efficiency, green chemi 

s t ry principle 6 (design for energy efficiency) 

clearly states, “If possible, synthetic 

methods should be conducted at ambient 

t e m p e r a t u re and pre s s u re.” While it can be 

argued that a separation method is not a 

synthesis method, the intent of using 

e x t remes of temperature and pre s s u re only 

when warranted is clearly implied. T h u s , 

no analysis of the greenness of SFC would 

be complete without addressing the issue of 

the high pre s s u re and temperature contro l


re q u i red with the use of supercritical carbon 

diox i d e . 

The critical point for pure carbon dioxide 

is 31.1°C and 73.8 bar, meaning that at 

t e m p e r a t u res and pre s s u res beyond these 

values, carbon dioxide exists as a superc r i t i- 

cal fluid. Clearly, any use of SFC must 

re s o rt to high pre s s u re, although only marginally 

higher than those addressable via 

s t a n d a rd pre p a r a t i ve HPLC pumping 

equipment. The major difference betwe e n 

SFC and HPLC instrumentation is the 

need for a back-pre s s u re regulator in SFC, 

whose function is to restrict outlet flow so 

as to create a back pre s s u re on the system, 

t h e re by maintaining the carbon diox i d e 

eluent in a supercritical or subcritical state. 

Thus, the need to operate carbon 

d i oxide–based pre p a r a t i ve chiral SFC at 

high pre s s u re does introduce some undesirable 

but unavoidable engineering and safety 

concerns that are more than adequately 

compensated for by the performance of the 

technique. 

When discussing carbon dioxide (a 

k n own greenhouse gas) as a green solve n t 

a l t e r n a t i ve, it is important to note that 

p re p a r a t i ve SFC is not a n e t generator of 

carbon dioxide. Instead it uses carbon dioxide 

that is condensed from the atmosphere 

or industrial waste plumes, shipped to the 

c h romatography installation, and then later 

returned to the atmosphere. In this re s p e c t , 

carbon dioxide is a re n ewable re s o u rce, and 

is a re c ove red industrial waste by p ro d u c t , 

both important green chemistry features. In 

contrast, incineration of waste organic solvents 

resulting from pre p a r a t i ve HPLC 

operations does result in the net generation 

of carbon diox i d e . 

Analytical Chiral SFC in Pharmaceutical 

Process Development 

We have long relied on SFC for carry i n g 

out analytical chiral separations, having 

found this technique typically to be faster 

and more convenient than HPLC separations 

(21–25). We employ an automated 

overnight column screening protocol using 

a standard gradient method for analytical 

method development, which usually afford s 

a usable method the next morning. Clearly, 

this approach offers a considerable adva n- 

tage over a manual one-at-a-time eva l u a t i o n 

of columns. When only one or a few analyses 

are re q u i red, the standard gradient 

method itself can be used without furt h e r 

modification. When analysis of more samples 

is re q u i red, conversion to an isocratic 

F i g u re 4: Commercial preparative SFC instruments currently being used in the authors’ 

l a b o r a t o r i e s . 

F i g u re 5: Representative chromatograms showing (a) a single injection and (b) repetitive stacked 

injections in kilogram-scale preparative SFC resolution of a diester intermediate. Columns: two 

270 mm 3 51 mm, 20-mm Chiralpak AD columns in series; mobile phase: 15% (mL/g) ethanol–carbon 

dioxide; flow rate: 350 g/min, pressure: 100 bar; temperature: 35 °C; detection wavelength: 

265 nm; racemate concentration: ;325 mg/mL; injection volume: 5 mL; cycle time: 235 s.


method often is performed to afford a 

s h o rter analysis time. For a single analytical 

i n s t rument, the green advantages of analytical 

SFC over analytical HPLC are slight in 

terms of waste solvent generation, although 

when one considers a large number of analytical 

instruments, the waste reduction is 

m o re substantial. 

S e m i p reparative Chiral SFC 

in Pharmaceutical Process 

D e v e l o p m e n t 

During the past few years, we have ro u- 

tinely used semipre p a r a t i ve SFC as our 

method of choice for rapid purification of 

small amounts of enantiopure materials, 

f rom a few milligrams up to about 20 g 

(11,25,26). Perhaps the most pro f o u n d 

a d vantage of semipre p a r a t i ve SFC purification 

comes from the fact that information 

obtained from the analytical column 

s c reening can be translated immediately 

into a workable pre p a r a t i ve SFC separation. 

This linking of analytical and pre p a r a t i ve 

techniques re q u i res matched pairs of analytical 

and pre p a r a t i ve columns, but this 

i n vestment allows one to re s o l ve gram 

amounts of completely unknown compounds 

within a day or two of receipt. T h e 

general advantage of SFC results in a considerable 

saving of time and materials, a 

savings that is advantageous from an economic 

standpoint, but also from a gre e n 

c h e m i s t ry perspective . 

The example depicted in Fi g u re 2 illustrates 

a typical use of semipre p a r a t i ve SFC 

in pharmaceutical development. Analytical 

SFC screening suggested Chiralcel OD 

(Chiral Technologies, Inc., West Chester, 

Pe n n s y l vania) was the best stationary phase 

for resolving the enantiomers of a deve l o p- 

mental compound. Immediate translation 

to a semipre p a r a t i ve SFC separation in isocratic 

mode was performed, and the sample 

load was increased to the point at which 

baseline resolution of enantiomers was just 

maintained. Repeated injections we re then 

p e rformed using an overlapping injection 

strategy to maximize the use of the column, 

ensuring that some compound was being 

eluted from the column at nearly all times. 

Separations of this type are ve ry conve n i e n t 

for preparing a few milligrams or a few 

grams of compound, and early access to 

JA N UA RY 2005 LCGC NORTH AMERICA VOLUME 23 NUMBER 1 2 7 

such material using the fast and labor-efficient 

tool of pre p a r a t i ve SFC can re vo l u- 

t i o n i ze the way that pharmaceutical 

re s e a rch is performed. In addition to providing 

material for testing synthetic ro u t e s 

or conducting pre l i m i n a ry biological eva l u- 

ations, the highly pure material obtained 

using semipre p a r a t i ve SFC can allow early 

re s e a rch into the critically important cry s- 

tallization experiments that often are used 

ultimately to effect purifications in pharmaceutical 

manufacturing at the industrial 

s c a l e . 

Kilogram-Scale Preparative Chiral 

SFC in Pharmaceutical Pro c e s s 

D e v e l o p m e n t 

Until re c e n t l y, chromatographic purification 

of amounts greater than about 20 g in 

these laboratories invo l ved the use of 

p re p a r a t i ve HPLC, which re q u i res deve l o p- 

ing a new chromatographic method or at 

least translation of an existing SFC method 

into an HPLC method. This translation 

p rocess is not always straightforw a rd. In 

some instances, developing a comparable 

H P LC separation for scale-up can prove

2 8 LCGC NORTH AMERICA VOLUME 23 NUMBER 1 JA N UA RY 2005 

difficult or impossible, and occasionally we 

found ourselves in the position of using a 

s e m i p re p a r a t i ve SFC instrument for carrying 

out a pre p a r a t i ve-scale separation. An 

example is shown in Fi g u re 3, which illustrates 

a resolution of enantiomers carried 

out at the .200-g scale that re q u i red thousands 

of injections and days of instru m e n t 

time to complete. Ne ve rtheless, automated 

sample injection and fraction-collection 

capability does make such separations re l a- 

t i vely straightforw a rd, although they are 

not as fast as one would like. 

It is at the kilogram scale that pre p a r a t i ve 

SFC begins to result in more dramatic savings 

in solvent, evaporation, and labor. 

Re c e n t l y, we acquired two large-scale 

p re p a r a t i ve SFC units (pictured in Fi g u re 

4), a 350-g/min system from Thar Te c h- 

nologies (Pittsburgh, Pe n n s y l vania), and a 

1-kg/min system from Novasep (Po m p e y, 

France). We have found this equipment to 

be quite helpful for performing the kilogram-scale 

purifications that are an essential 

component of modern pharmaceutical 

d e ve l o p m e n t . 

An example aptly illustrates the value of 

the SFC approach. We we re called upon 

recently to re s o l ve the enantiomers of a 

pharmaceutical intermediate not re a d i l y 

accessible via enantioselective synthesis. In i- 

tial screening followed by method optimization 

using loading studies led to optim 

i zed methods in both the SFC and 

H P LC modes. Although optimized SFC 

methods for chiral separation often are 

m o re pro d u c t i ve than the corre s p o n d i n g 

o p t i m i zed HPLC method, the SFC adva n- 

tage can be quite profound. Such was the 

case in the current example, where we 

o b s e rved a roughly 10-fold greater pro d u c- 

tivity for the SFC method re l a t i ve to the 

H P LC method. It should be pointed out 

that both methods we re fully optimized to 

the best of our abilities, and although the 

SFC advantage in productivity might more 

typically be in the range of twofold, much 

larger productivity advantages are by no 

means uncommon. In this part i c u l a r 

instance, we we re presented with the choice 

of performing a separation using either a 

30-cm i.d. HPLC column (36,000 L of solvent, 

10,000 L evaporated) or a 5-cm i.d. 

SFC column (900 L solvent, 215 L eva p o- 

rated), with instrument time projected to 

be about 120 h for either approach. T h e 

choice in this instance was obvious, and 

SFC was used to carry out the separation. 

A re p re s e n t a t i ve chromatogram (Fi g u re 5) 

illustrates a single injection of 1.5 g of racemate 

and a series of overlapping injections 

re p re s e n t a t i ve of the actual campaign. 

A second case is illustrated by an example 

in which there was a need to purify at 

least 1.5 kg of a single diastereomer of an 

i n vestigational compound. In this instance, 

d i f f e rences between the optimized HPLC 

and SFC methods we re even more profound, 

as no suitable pre p a r a t i ve HPLC 

separation could be discove red. The separation 

was performed using pre p a r a t i ve SFC, 

and 1.725 kg of purified diastere o m e r 

( 9 9 % d i a s t e romeric excess) was obtained in 

about 72 h using a 5-cm i.d. Chiralpak AS 

column (Chiral Technologies) with an eluent 

of 30% isopropanol in carbon diox i d e 

(100 bar outlet pre s s u re, 350 g/min) and 

using about 830 L of solvent (275 L eva p o- 

rated). These results illustrate the utility of 

p re p a r a t i ve SFC for generating kilogram 

amounts of development compounds and 

e m p h a s i ze the tremendous saving in 

organic solvent use that can be possible 

with pre p a r a t i ve chiral SFC. 

Recent experience 

with larger scale SFC 

systems has shown 

that the SFC 

advantage can be 

quite useful for 

p roviding purified 

materials on the 

kilogram scale. 

P reparative SFC in Pharmaceutical 

P rocess Development: Future 

P ro s p e c t s 

Separation of amounts greatly in excess of a 

f ew kilograms would re q u i re the need for 

e ven larger pre p a r a t i ve SFC units than 

those pictured in Fi g u re 4. In t e re s t i n g l y, the 

relationship between pre s s u re and vo l u m e 

places some constraints for the scale-up of 

p re p a r a t i ve SFC columns. As column diameter 

increases, the need for eve r - i n c re a s i n g 

wall thickness can place a practical and economic 

limit on the scalability of a stainless 

steel column construction approach. Howe 

ve r, industrial scale supercritical carbon 

d i oxide extraction vessels (see, for example, 

w w w.uhde-hpt.com) in the 1-m i.d. range 

and operating at much higher pre s s u re s , 

t e m p e r a t u res, and flow rates (for example, 

600 bar, 80 °C, 20 kg/min) than those typically 

used in pre p a r a t i ve chiral SFC, suggest 

that construction of the next size increment 

of pre p a r a t i ve SFC units should be 

feasible technologically. An intere s t i n g 

a p p roach to ove rcoming the pre s s u re / vo l- 

ume problem at larger scale is the use of a 

multicolumn SFC approach, in which the 

adsorbent is spread between a number of 

columns of manageable dimensions. Pro t o- 

type SFC-SMB systems have been 

described by Novasep (27) and Jo h a n n s e n 

and colleagues (28,29) and show some 

p romise for separations at industrial scale. 

In t e re s t i n g l y, the SFC-SMB approach offers 

the promise of more efficient desorption of 

the more retained enantiomer simply by 

i n c reasing the pre s s u re in the desorption 

zone of the SMB, an approach that could 

f u rther improve perf o r m a n c e . 

C o n c l u s i o n 

In summary, we have found SFC to be an 

i m p o rtant tool with many green chemistry 

a d vantages for supporting preclinical deve l- 

opment in the pharmaceutical industry. 

Our previous experience had shown that 

p re p a r a t i ve SFC was the method of choice 

for rapidly accessing pure and enantiopure 

materials on a scale up to a few grams. 

Recent experience with larger scale SFC 

systems has shown that the SFC adva n t a g e 

can be quite useful for providing purified 

materials on the kilogram scale, where the 

g reen chemistry advantages of solvent and 

waste reduction make the approach attract 

i ve from both an economic and enviro n- 

mental standpoint. 

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K. Hansen, P.N. Devine, P.J. Pye, K. Ro s s e n , 

P.G. Do r m e r, R.A. Re a m e r, C.J. Welch, D.J. 

Ma t h re, N.N. Tsou, J.M. Mc Namara, and P. J . 

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Ma t h re, C h i ra l i t y 1 5, 143–147 (2003). 

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and D.J. Ma t h re, C h i ra l i t y 15, 609–613 (2004). 

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Metabolism & Disposition 32, 1061–1068 

( 2004 ). 

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( 2004 ). 

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JA N UA RY 2005 LCGC NORTH AMERICA VOLUME 23 NUMBER 1 2 9 

( 1 9 9 8 ) . 

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Biochem. Biophys. Me t h . 5 4(1–3), 85–102 

( 2 0 0 2 ) . 

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n e r, J. Chro m a t o g r., A 8 6 5(1–2), 175–186 

( 1 9 9 9 ) . 

Christopher J. Welch heads the Analysis and 

Preparative Separations Group in the Process 

Research Department at Merck & Co., Inc., 

R a h w a y, NJ, and has more than 20 years of 

experience in the analytical and preparative 

chromatographic separation of enantiomers.

Column Wa t c h C o l u m n - Pharmaceutical Technology

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