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Barnhart, Gahm, Tomas, Notari, Semin, Cheetham 619

Wesley W. Barnhart Supercritical fluid chromatography tandem-column
Kyung H. Gahm
Sam Thomas method development in pharmaceutical sciences
Steve Notari for a mixture of four stereoisomers
David Semin
Janet Cheetham
A tandem-column method using Chiralpak AD-H and Chiralcel OD-H columns was
Discovery Analytical Sciences, achieved for baseline separation of a mixture of chiral pharmaceutical compounds
Molecular Structure, Amgen Inc., (i. e., four stereoisomers) via supercritical fluid chromatography (SFC) with a mobile
Thousand Oaks, CA 91320, USA phase consisting of 90% liquid carbon dioxide and 10% ethanol:isopropanol (50 : 50
v/v). On the contrary, this mixture (mixture A) could not be baseline separated by
SFC conditions explored with individual Chiralpak AD-H and Chiralcel OD-H columns.
The effects of various mobile phases on elution order, capacity factor, selectivity, and
resolution were determined with mixture A on the individual aforementioned columns
to develop the tandem-column method.
Key Words: Supercritical fluid chromatography; Tandem-column; Diastereomer;
Received: January 4, 2005; revised: February 15, 2005; accepted: February 24, 2005
DOI 10.1002/jssc.200500005

1 Introduction can be overcome is to utilize tandem columns to obtain a
desired single-method separation. In this paper, an analy-
Supercritical fluid chromatography (SFC) is an important tical SFC method was developed by employing the cou-
analytical and preparative tool used in the pharmaceutical pling of Chiralpak AD-H and Chiralcel OD-H columns to
industry. Separation of chiral pharmaceutical compounds separate a mixture of pharmaceutical compounds (i.e.,
is an ever-increasing chromatographic field [1], and chiral- four stereoisomers); this separation was not achieved
ity is an important factor in drug development [2 – 5] due to using a single-column method. By understanding the elu-
the chiral specificity of biological processes [6]. Separa- tion order and effects of various mobile phases on the
tion of chiral compounds is vital because the activity of separation of the compounds by Chiralpak AD-H and
each enantiomer must be explored; it is possible that one Chiralcel OD-H columns separately, the development of
enantiomer may be inactive, an antagonist, or toxic [5 – 7]. the tandem-column method was made possible. A single-
A classic example is thalidomide. Originally marketed as a method separation is more desirable than multiple meth-
sedative, thalidomide was a racemic mixture; one enantio- ods, since it is less time consuming and cumbersome,

Original Paper
mer was a sedative, while the other was teratogenic [8, 9]. requiring no change of column or mobile phase conditions
SFC was the new and exciting chiral chromatographic between the analyses of different compounds.
topic in the early to mid 1980s [10, 11] when it was demon-
The use of coupled columns to separate compounds chro-
strated to be a technique capable of separating enantio-
matographically is not a novel concept [19, 22 – 27]; the
mers [12]. The use of SFC for the separation of enantio-
difficulty, however, is the determination of the correct
mers has been one of its most successful applica-
combination of columns given the large number of station-
tions [13]. Though the technique is over 20 years old, its
ary phases available on the market. According to San-
potential is continuing to be explored. SFC is a popular
dra [23], the selectivity and efficiency of a single column
chiral separation technique due to the inherent advan-
often does not provide adequate separation of a given
tages of using liquid CO2 in the mobile phase [1, 3, 11,
mixture of compounds. Recently, Phinney et al. [24]
14 – 21].
coupled achiral and chiral columns to separate a mixture
The development of a successful and facile single-column of achiral and chiral compounds.
analytical chiral separation method is not always achieved For our study, however, two chiral columns were coupled
due to limited in-house column selections or time restric- for the purpose of efficiently separating chiral compounds
tions imposed by the project cycle times of early discovery (a mixture of four stereoisomers). The initial choice to
chemistry projects. One way in which these limitations screen the Chiralpak AD-H and Chiralcel OD-H columns
was based on past successful experiences with these col-
Correspondence: Wesley W. Barnhart, Discovery Analytical
Sciences, Molecular Structure, Amgen Inc., Thousand Oaks, CA umns; Chiralpak AD and Chiralcel OD columns have been
91320, USA. Phone: +1 805 447 2055. Fax: +1 805 480 3015. shown to be very successful and widely used in chiral
E-mail: wesleyb@amgen.com. chromatography [1, 28, 29]. In addition to separating four

J. Sep. Sci. 2005, 28, 619 – 626 www.jss-journal.de i 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

620 Barnhart, Gahm, Tomas, Notari, Semin, Cheetham

stereoisomers in a single method analytically, the mixture variable wavelength detector, and a waste containment
was also separated preparatively. As a proof of concept, vessel. The injector was a Modular Digital Pump (Model
approximately 1 mg of the four-stereoisomer mixture was XL3000) from Cavro Scientific Instruments Inc. (Sunny-
separated to baseline. The two coupled columns, though vale, CA, USA). The software used in the purification was
slightly cumbersome, did fit neatly into the column oven Berger SFC ProNTo v. 1.5.305.15.
heater and allowed the separation of all four stereo-
isomers in a single method. 2.4 Chiral packed columns
The analytical chiral packed columns were Chiralpak AD-
2 Experimental H and Chiralcel OD-H. Columns were purchased from
Chiral Technologies (Exton, PA, USA). The columns are
2.1 Materials
referred to as AD-H and OD-H throughout the paper.
The SFC-grade carbon dioxide was obtained from BOC Dimensions of the columns were 15064.6 mm ID with
Gases (Murray Hill, NJ, USA). Methanol (MeOH), ethanol 5 lm particle size.
(EtOH), and isopropanol (IsOH) were HPLC-grade from
Mallinckrodt Baker (Muskegon, MI, USA). The 1-propanol For preparative SFC, the chiral packed columns consisted
(nPrOH) and 1-butanol (nBuOH) were purchased from of Chiralpak AD-H (250621 mm, 5 lm) and Chiralcel
Sigma-Aldrich (St. Louis, MO, USA). All aforementioned OD-H (250621 mm, 5 lm) columns. Columns were pur-
solvents were of analytical grade. chased from Chiral Technologies (Exton, PA, USA). The
preparative columns are referred to as prep-ADH and
The compounds were synthesized in-house and dis- prep-ODH throughout the paper.
solved in methanol for analysis. The compound mixture
consisted of four stereoisomers and will be referred to
2.5 Analytical analysis conditions and
hereafter as mixture A. Individual stereoisomers of mix-
calculations
ture A were obtained by multi-step purification prior to the
development of the single-step, tandem-column prepara- For single-column analytical analyses, the mobile phase
tive method. The single-step purification method was consisted of 90% liquid CO2 and 10% organic modifier.
developed for the possibility of future purifications and to Organic modifiers were MeOH, EtOH, IsOH, nPrOH, and
provide a more efficient purification process than the nBuOH. For tandem-column analyses, the primary col-
multi-step method. umn order was AD-H and OD-H, respectively. These col-
umns were connected by a two-inch piece of stainless
2.2 Analytical SFC instrumentation steel tubing. Organic modifiers were EtOH, IsOH, and
EtOH:IsOH (50 : 50 v/v). Methods were isocratic with a
The analytical SFC instrument was a Berger SFC unit flow rate of 3.0 mL/min. Column oven and nozzle tem-
(Mettler-Toledo Autochem, Newark, DE, USA) with an perature were 408C, and the outlet pressure was 120 bar.
FCM1200 flow control module, a dual pump control mod-
ule, a TCM2100 thermal column module (temperature is Retention times used for calculating capacity factor and
controlled from 7 – 1508C), a column selection valve cap- resolution were obtained from the chromatographic data
able of switching between six columns, and a solvent con- generated via MassLynx v. 4.0 SP1. Void volume was
trol valve permitting selection of up to six modifiers. The estimated by using the retention time of the peak that
SFC was equipped with an Agilent 1100 photodiode array resulted from the change in refractive index from the injec-
detector with a high-pressure flow cell (Agilent Technolo- tion solvent. Peak widths were measured manually.
gies, Palo Alto, CA, USA). The autosampler/injector was
a CTC LC Mini PAL from Leap Technologies (Carrboro, 2.6 Preparative analysis conditions
NC, USA). A Waters (Milford, MA, USA) ZQ benchtop sin- The mobile phase consisted of 90% liquid CO and 10%
2

gle quadrupole mass spectrometer with an atmospheric EtOH:IsOH (50 : 50 v/v). The method was isocratic with a
pressure chemical ionization (APCI) source was coupled flow rate of 55 mL/min. Column oven and nozzle tempera-
to the SFC. The software packages used in the analyses ture were 408C, and the outlet pressure was 120 bar.
were Berger MassWare v. 4.01 and MassLynx v. 4.0 SP1.

3 Results and discussion
2.3 Preparative instrumentation
The preparative SFC was a Berger Multigram II from Met- 3.1 Single column analyses of mixture A
tler-Toledo Autochem (Newark, DE, USA). The compo- To gain a better understanding of the effects of organic
nents were the Separator Control Module (SCM)-2500, modifiers (i. e., MeOH, EtOH, IsOH, nPrOH, and nBuOH)
Electronics Control Module (ECM)-2500, CO2 solvent on the separation of the four stereoisomers, mixture A
delivery module, modifier solvent delivery module, direct was screened overnight with the OD-H and AD-H columns
expansion probe chiller, ventilated collection cabinet, UV separately (Figure 1 and Figure 2, respectively). The


SFC tandem-column method development 621

Figure 1. Analytical chromatograms of mixture A with the OD-H column. The mobile phase consisted of 90% liquid CO2 and 10%
of either MeOH, EtOH, IsOH, nPrOH, or nBuOH at a flow rate of 3.0 mL/min. Column temperature and outlet pressure were
408C and 120 bar, respectively. Peaks 1 and 4 are an enantiomeric pair (S,R and R,S), and Peaks 2 and 3 are an enantiomeric
pair (S,S and R,R). (Peak number indicates and correlates the order of elution with the OD-H column.)

goal was to develop a single method to separate all four with EtOH and nBuOH used as the organic modifer. Over-
stereoisomers. No single-column method, however, all, however, the elution order was not predictable with
yielded baseline separation of all four stereoisomers. respect to the various organic modifiers that were studied.
Therefore, the individual stereoisomers (designated as A closer look at individual peaks and their elution order
Peaks 1 through 4) were screened by OD-H (Figure 1) with respect to modifier (Figure 2) indicated three different
and AD-H (Figure 2) columns to determine the identity of cases. One such case was the elution order of Peak 2. Its
the peaks noted in the screening of mixture A. The stereo- elution order remained the most varied throughout the use
chemistry of the stereoisomers represented by Peaks 1, of the various modifiers. When MeOH was used as the
2, 3, and 4 are S,R; S,S; R,R; and R,S, respectively. The modifier, Peak 2 eluted first. With IsOH as the modifier,
peak number is based on the peak order found with the though, Peak 2 eluted last. Only with EtOH and nBuOH (2
OD-H column, since the peak order remained constant out of 5 modifiers) did Peak 2 have a consistent elution
regardless of the modifier. order. The second case is the elution of Peak 1. Peak 1
Considering the OD-H column results (Figure 1), no remained most consistent with its elution order. Except
change in peak order was observed throughout the use of when MeOH was used as the modifier, Peak 1 eluted first
the various mobile phases. Overall, adequate separation (4 out of 5 modifiers). Lastly, the elution order of Peaks 3
of all four components was not achieved with the OD-H and 4 remained the same for three of the five modifiers
column. Further exploration with IsOH via the OD-H col- (MeOH, EtOH, and nBuOH). Overall, as observed with
umn was performed, but baseline separation of Peaks 2 the OD-H column, no single AD-H method provided ade-
and 3 was not achieved. Decreasing the amount of IsOH quate separation of the four stereoisomers.
in the mobile phase did not improve separation.
Figure 2 clearly shows the elution order varied for Peaks 1 3.2 Capacity factor (k 9) of individual peaks of
through 4 via the AD-H column with the use of different mixture A for OD-H and AD-H columns
organic modifiers in the mobile phase. The only two ana- The capacity factor (k 9) for the four individual stereo-
lyses that yielded the same order of elution (1-2-4-3) were isomers was calculated and plotted (Figure 3). When



Figure 2. Analytical chromatograms of mixture A with the AD-H column. The mobile phase consisted of 90% liquid CO2 and 10%
of either MeOH, EtOH, IsOH, nPrOH, or nBuOH at a flow rate of 3.0 mL/min. Column temperature and outlet pressure were
408C and 120 bar, respectively. Peaks 1 and 4 are an enantiomeric pair (S,R and R,S), and Peaks 2 and 3 are an enantiomeric
pair (S,S and R,R). (Peak number indicates and correlates the order of elution with the OD-H column.)

comparing k 9 values for Peaks 1 through 4 for both the
OD-H and AD-H columns, it appears that there was a
clear difference between columns in the trend found for k 9.
The k 9 values of the AD-H column with nBuOH (7.2, 9.4,
11.5, and 10.0 for Peaks 1, 2, 3, and 4, respectively) were
greater than the OD-H column with nBuOH (4.5, 5.8, 6.1,
and 8.3 for Peaks 1, 2, 3, and 4, respectively). However,
IsOH provided the largest k 9 values (i. e., 12.7 and 22.0 for
Peak 4 via AD-H and OD-H, respectively) with both col-
umns.

All four stereoisomers showed the same trend with the
OD-H column. The k 9 increased from MeOH to IsOH, with
the maximum value (8.0, 11.2, 13.3, and 22.0 for Peaks 1,
2, 3, and 4, respectively) found using IsOH as the modi-
fier. Unlike the AD-H column results, Peaks 2 and 3 main-
tained the most similar k 9 values between the use of the
various organic modifiers with the OD-H column.

For the AD-H column, all of the peaks did not follow the
same pattern with respect to the k 9 values; however, simi-
larities did emerge. Peaks 1 and 3 had very similar pat-
terns of k 9 vs. modifier (Figure 3). Also, Peaks 2 and 4
Figure 3. Capacity factor (k9) of all four peaks with various
showed similar trends and k 9 values. Peaks 2, 3, and 4 mobile phase components utilizing OD-H and AD-H col-
show very similar results from IsOH through nBuOH as umns. Liquid CO2 is 90% of the mobile phase, with the
the modifier. Both the pattern and k 9 values are very simi- remaining 10% as MeOH, EtOH, IsOH, nPrOH, or nBuOH.



3.4 Analytical scale AD-H-OD-H tandem column
analyses of mixture A
Individual AD-H and OD-H columns did not provide an
adequate method for the baseline separation of all four
stereoisomers. The individual column results previously
discussed, however, indicated the possibility of separat-
ing all four stereoiosomers if the AD-H and OD-H columns
were coupled. Organic modifiers were chosen based on
the results of the individual AD-H and OD-H column
screening. When considering tandem-column LC, the
retention time noted for a particular analyte is the sum of
the retention times of the analyte for the individual col-
umns; this is independent of the column order [25]. It was
by adding the peak retention times for the individual AD-H
and OD-H columns (with EtOH and IsOH as the organic
modifiers) that there appeared the possibility of resolving
all four stereoisomers through the coupling of the two col-
umns.
The tandem-column methods explored utilized mobile
phases that consisted of 90% liquid CO2 and 10% EtOH,
IsOH, or EtOH : IsOH (50 : 50 v/v) (Figure 5). Looking at
Figure 4. Resolution of Peaks 1 & 4 (D) and 2 & 3 (F) utilizing
different mobile phase components (MeOH, EtOH, IsOH, the tandem-column separation with EtOH and IsOH indivi-
nPrOH, and nBuOH) with the OD-H and AD-H columns. dually, it was predicted that a combination of the two
Liquid CO2 is 90% of the mobile phase, with the modifier would provide the desired separation. A combination of
(MeOH, EtOH, IsOH, nPrOH, or nBuOH) as the remaining EtOH and IsOH (EtOH : IsOH (50 : 50 v/v)) was then used
10%.
to achieve the separation of the four stereoisomers with
an analysis time of less than 15 min. Since baseline sep-
lar, indicating the AD-H is a column that had very similar
aration was already achieved with the 50 : 50 mixture,
affinities for all three peaks with IsOH, nPrOH, and
further exploration of EtOH : IsOH combinations for
nBuOH as the modifiers.
method optimization was not conducted. Also, reversing
the order of the tandem-columns did not result in a notable
difference in the separation of the stereoisomers.

3.3 Resolution (R ) of enantiomeric pairs of By utilizing the separation characteristics of individual
mixture A for OD-H and AD-H columns Chiralpak AD-H and Chiralcel OD-H columns, it was pos-
sible to obtain baseline separation of all four of the stereo-
The resolution (R) for the enantiomeric pairs was calcu- isomers through the coupling of the two columns; this was
lated and plotted (Figure 4). The resolution plots in Fig- not achieved with a single column. In addition, the base-
ure 4 show similar patterns found for the selectivities (not line separation was only accomplished by combining two
shown), as expected. For the OD-H column, the resolution mobile phase components (EtOH and IsOH) for the
patterns of both peak pairs (Peaks 1 & 4 and 2 & 3) were coupled column method.
very similar; however, Peaks 1 & 4 demonstrated much
greater R values than those found with Peaks 2 & 3, thus
indicating that the OD-H column was much better suited 3.5 Application of the analytical tandem-column
for separating the enantiomers 1 & 4 than 2 & 3. Peaks 1 & method
4 were best separated on the OD-H column using MeOH The analytical tandem-column SFC method was success-
as the organic modifier. fully applied to quickly determine the results of a reaction
A closer look at the AD-H results showed that each of the designed to transform a precursor into two of the four
enantiomeric pairs had much different behavior. The stereoisomers found in mixture A. Figure 6 shows the
modifier that resulted in the greatest R value for enantio- chromatograms indicating the precursor, the two stereo-
meric pairs 2 & 3 was MeOH. The lowest R value for enan- isomers formed, and a chromatogram of the original mix-
tiomeric pairs 2 & 3 was obtained through the use of IsOH ture A. The precursor could be easily separated from the
as the organic modifier. For 1 & 4, the greatest R value stereoisomers in mixture A, which would provide a quick
was observed with nPrOH as the modifier, and the lowest and facile procedure to monitor both the presence of the
R value resulted from the use of MeOH as the modifier. precursor and the product of the reaction utilizing a single



Figure 5. Chromatograms of the AD-H and OD-H column coupling for the separation of the four stereoisomers. The mobile
phase consisted of 90% liquid CO2 and 10% EtOH, IsOH, or EtOH : IsOH (50 : 50 v/v). The temperature and outlet pressure were
408C and 120 bar, respectively. Total flow rate was 3.0 mL/min. (Peak number indicates and correlates the order of elution with
the OD-H column.)

method. This was critical for a timely analysis of the single-column method. However, with the columns in tan-
results of the reaction. A single column could have been dem, a single method was successfully established. The
employed to resolve Peaks 1 and 2; however, both benefit of a single method was quickly realized when a
Peaks 3 and 4 would not have been identified (i. e., reaction (that converted a precursor into two of the stereo-
resolved) if they had also been formed during the reac- isomers of mixture A) was efficiently and effectively moni-
tion. tored; this provided critical data in a timely manner to
easily fit within the timelines provided for project support.
3.6 Preparative scale AD-H-OD-H tandem-column Overall, the order of elution of the four stereoisomers with
separation of mixture A the AD-H column was difficult to predict, while the order of
After establishing a tandem-column analytical method, elution remained constant with the OD-H column. This
the next step was to attempt a preparative separation. To indicates the separation mechanism on the AD-H column
demonstrate proof-of-concept, the same mobile phase for these compounds is more complicated compared to
composition (90% liquid CO2 and 10% EtOH : IsOH the OD-H column. Since both columns contain the same
(50 : 50 v/v)) was used for the preparative separation (Fig- derivative (tris-3,5-dimethylphenylcarbamate [30, 31]),
ure 7) as that used on the analytical scale. The overall the behavior of the stereoisomers with the AD-H column is
separation time was 24 min. This example illustrates how most likely due to the difference in the structure of amy-
a single method with coupled columns can be used to lose (AD-H) compared to cellulose (OD-H). The helical
baseline separate four stereoisomers and facilitate the nature of amylose allows for better inclusion when com-
purification process. pared to the more planar cellulose [3]. With the AD-H col-
umn, different alcohol modifiers in the mobile phase can
alter the chiral cavities and could modify the elution order
4 Conclusions of the peaks [32]. The AD-H column may therefore allow
Method development to separate a mixture of four stereo- more diverse interactions than the OD-H column resulting
isomers with AD-H or OD-H columns did not produce a in varied elution order with change of organic modifier.



Figure 6. Application of the tandem-column method for the analysis of the end products of a reaction. Chromatograms of a pre-
cursor, peaks 1 & 2, and mixture A are shown. The mobile phase consisted of 90% liquid CO2 and 10% EtOH : IsOH (50 : 50 v/v).
The temperature and outlet pressure were 408C and 120 bar, respectively. Total flow rate was 3.0 mL/min. (Peak number indi-
cates and correlates the order of elution with the OD-H column.)

Figure 7. Preparative chroma-
togram of 1 mg of mixture A uti-
lizing the tandem-column
method. The mobile phase con-
sisted of 90% liquid CO2 and
10% EtOH : IsOH (50 : 50 v/v).
The column oven and nozzle
temperature were 408C, and
the outlet pressure was
120 bar. Total flow rate was
55 mL/min. (Peak number indi-
cates and correlates the order
of elution with the OD-H col-
umn.)

An advantage of analyzing the effects of organic modifiers References
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