1

Wat ers solut ionsPrep 100 SFC MS Directed System

2767 Sample Manager

3100 Mass Detector

2998 Photodiode Array Detector (PDA)

Gas-Liquid Separator

MassLynxTM Software

K eyWordsOpen-bed fraction collection

CO2 expansion

SFC

Purification

Mass-Directed Preparative SFC with Open-Bed Fraction CollectionRui Chen, Ph.D. Waters Corporation, USA

int roduCt ion In recent years, improving productivity and cost-savings have become major initiatives

in pharmaceutical discovery and development. The introduction of new technologies

for high-throughput synthesis has enabled preparation of up to 48 compounds in the

same time frame previously required for a single compound1. Consequently, there has

been increasing pressure on development and implementation of efficient purification

platforms to support the overall high throughput process.

Mass-directed reverse phase liquid chromatography (RPLC) purification has revolution-

ized the workflow for chemists in drug discovery by enabling one fraction collection

per injection, largely due to the specificity that MS detection offers. As a result,

mass-directed RPLC has quickly become the most commonly used technique in high

throughput purification environments. Considerable effort has been put into stream-

lining the process so that minimal user intervention is required. However, a major

limitation in the process is the long dry-down time of aqueous fractions downstream.

Adopted primarily for chiral analysis and purification by the pharmaceutical indus-

try in the early 1990s, supercritical fluid chromatography (SFC) has experienced

a remarkable resurgence in the past decade. The reduction in solvent consumption

and collection in relatively small volumes of volatile organic solvents in preparative

SFC has led to significant savings on operational costs. For example, Ripka et al.

calculated that 20,000 samples purified by SFC instead of RPLC would realize a

48 times reduction in solvent consumption2.

A mass-directed preparative SFC system, like its counterpart in RPLC, is an ideal platform

for high-throughput purification of diverse libraries of drug-like compounds. However,

open-bed fraction collection has been a challenge for SFC due to aerosol formation

caused by the depressurization of CO2, particularly at high flow rates. Kassel et al.

demonstrated the feasibility of mass-triggered SFC fraction collection at 15 mL/min3 and

Zhang et al. reported their results with flow rates up to 30 mL/min4. The challenge of

managing aerosols in collection at high flow rates has been overcome with the develop-

ment of Waters® Prep 100 SFC MS Directed System5.

In this application note, we present our research results to illustrate one of the

key innovations of the Prep100 SFC MS Directed System: the Gas-Liquid Separator

(GLS). Some comparative results on library compound purification using both

AutoPurification® LC/MS and Prep 100 SFC MS Directed systems are also presented

to highlight the orthogonality of the two techniques.

ap pliCat ion benefits The Gas-Liquid Separator (GLS) in the Prep 100

SFC MS Directed System enables open-bed fraction

collection at high flow rates. Combined with the

AutoPurification LC MS system in high throughput

purification environments, the orthoganility

between RPLC and SFC offers potential cost-savings

by recovering more compounds from medicinal

chemistry for ensuing research and development.

2 Mass-Directed Preparative SFC with Open-Bed Fraction Collection

Figure 1. Schematic of the Prep 100 SFC MS Directed System.

syst eM ConfiGurat ionAll preparative experiments were performed on a Prep 100 SFC MS Directed System,

as shown in Figure 1, which consists of: Waters 2767 Sample Manager, 3100 MS

Detector, 2998 PDA Detector, 515 Pumps and Waters CO2 Pump and Co-solvent

Pump, Analytical-2-Prep Column Oven, Automated Back Pressure Regulator

(ABPR), GLS, and Tunable Splitter. The system was controlled by Waters

MassLynx™ Software.

results and disCussionOne of the major challenges in implementing open-bed fraction collection at high

flow rates in SFC is CO2 expansion. After exiting the ABPR, high pressure liquid

CO2 decompresses to its gaseous form at a volume ratio of approximately 1:500.

This rapid expansion causes aerosol formation, which can lead to sample loss and

cross contamination. This challenge is addressed by a proprietary GLS, as shown in

Figure 2. The geometry of the GLS, the dimension of the initiator, and its angle with

respect to the inner wall effectively eliminate the majority of gaseous CO2. Another

key aspect of the GLS implementation is to preserve the peak integrity during a

Figure 2. Schematic of the GLS.

3 Mass-Directed Preparative SFC with Open-Bed Fraction Collection

gradient before and after the GLS, such that time delay between detectors and collector tip across a gradient can

be accounted for correctly. To accomplish that, a make-up flow of methanol with a reverse gradient is introduced

to ensure a constant flow passing through the GLS, especially at the point in the gradient when the co-solvent per-

centage is low. To prove the efficiency of the GLS, two detectors, one placed in the main flow path before the GLS

and the other after the GLS in place of the collector tip, were used to compare the pre- and post-GLS peak profiles.

Figure 3 shows the two traces at different modifier percentages from the two UV detectors. It is evident that peak

profiles were well maintained passing through the GLS. The maximum band broadening due to GLS is 1.2 s (results

not shown) at the peak tail, representing a less than 2% sample loss.

10% Co-solvent 30% Co-solvent 50% Co-solvent

The effectiveness of the GLS is demonstrated by the high recovery and high purity of collected fractions. For example,

Aurigemma et al. reported their evaluation of the Prep 100 SFC MS Directed System5 . In their experiment, a mixture

containing 8 mg/mL each of (A) flavone, (B) carbamazepine, (C) acetaminophen, and (D) sulfamethazine was injected

and collected over three replicate injections, as shown in Table 1. Overall, sample recovery and purity was greater

than 95% (with the exception of peak C in injection 1) during gradient elution. In particular, the high purity of two

closely eluting components (C and D) indicated a negligible cross contamination between peaks.

In a separate experiment6, similar evaluations were performed using a mixture of caffeine, sulconazole, and

bendroflumethazide. On average, greater than 90% recovery and greater than 99% purity were achieved for

all compounds using UV triggering, MS triggering, and UV+MS triggering, respectively.

An additional built-in advantage of the Prep 100 SFC MS Directed System is the software continuity from the

analytical platform, Resolution™ SFC MS System, capable of both pre-purification scouting and post-purification

analysis. Both platforms are controlled by the MassLynx Software package. After the analytical scouting run,

data is processed and the retention time for the ion of interest is used to match one of the pre-defined focused

gradients on the preparative system. The purification strategy is automatically determined and exported to the

preparative platform. The whole process is automated by AutoLynx™ and FractionLynx™ application managers,

part of MassLynx Software control.1 Furthermore, since both Prep 100 SFC MS Directed and AutoPurification

LC/MS systems are controlled by MassLynx Software, it should be effortless for existing AutoPurification

LC/MS users to adapt Prep 100 SFC MS Directed into their workflow.

UV

MS triggered collection portion

Collector tip

Same portion at collector tip

Figure 3. UV traces from two detectors at 10%, 30%, and 50% co-solvent.

4 Mass-Directed Preparative SFC with Open-Bed Fraction Collection

inj. peak uV purity tiC purity recovery (tiC)

A 100.0 100.0 99.0

B 100.0 100.0 97.0

C 100.0 100.0 86.0

D 98.7 99.1 98.0

A 100.0 100.0 97.0

B 100.0 100.0 96.0

C 100.0 100.0 97.0

D 100.0 99.2 97.0

A 100.0 100.0 97.0

B 100.0 99.5 95.0

C 100.0 100.0 99.0

D 100.0 99.4 98.0

Implementing the Prep 100 SFC MS Directed System into high-throughput purification environments provides a

cost-savings that results from reduction in solvent consumption and dry-down time2,7. In addition, the orthogonal-

ity between SFC and RPLC offers increased cost-savings by pushing more compounds from medicinal chemistry

through the pipeline. SFC is loosely considered a normal phase chromatographic technique and complements RPLC

in selectivity. For example, Figure 4 shows the comparison between mass-directed RPLC and SFC in purifying a

real-world pharmaceutical compound directly from combinatorial synthesis; in which, the impurity was resolved

from the target compound in SFC but not in RPLC. In our previous study8, we reported a 30% increase in success

rate when combining the two techniques compared to using each technique alone. In the same study, we also

reported a 20% reduction in overall batch processing time for SFC compared to RPLC as a result of shorter dry-

down time post-purification.

Figure 4. RPLC and SFC chromatograms illustrating the different selectivity between RPLC and SFC.

i

njec

tion

1

inje

ctio

n 2

in

ject

ion

3

Table 1. Purities and recoveries of SFC collections.

0

100

0 1 2 3

Inte

nsity

(%)

Time (min)

0%

100%

130 207 251 323 354m/z

RPLC

321

349

0

20

40

60

80

100

0 1 2 3

Ab

so

rba

nc

e (

AU

)

Time (min)

0

100

0 2 4 6 8

Inte

nsity

(%)

Time (min)

0%

100%

125 187 320 395m/z

0%

100%

130 206 323 377m/z

SFC

349 321

0

100

200

300

400

500

0 2 4 6 8

Ab

so

rban

ce (

AU

)

Time (min)

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Waters and AutoPurification are registered trademarks of Waters Corporation. The Science of What’s Possible, Prep 100, MassLynx, Resolution, AutoLynx, and FractionLynx are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2010 Waters Corporation. Produced in the U.S.A.May 2010 720003468en AG-PDF

ConClusionsn The GLS, the key innovation in the Prep 100 SFC MS Directed System, has been able

to overcome one of the main historical challenges in SFC open-bed fraction collection

n Greater than 90% recovery and greater than 95% purity were achieved in two

independent evaluations, which indicates the effectiveness of the GLS

n The level of automation available through the software between the analytical

and preparative platforms enables a streamlined analytical to purification

process with minimal user intervention

n The orthogonality between mass-directed RPLC and SFC can potentially offer

users additional cost-savings by recovering more compounds from medicinal

chemistry for ensuing research and development

references:

1. McClain, RT et al., Journal of Liquid Chromatography & Related Technologies, 2009; 32: 483–499.

2. Ripka, WC, Barker, G, and Krakover, J., Drug Discovery Today, 2001; 6(9): 471-477.

3. Wang, T. et al., Rapid Commun. Mass Spectrom, 2001; 15: 2067-2075.

4. Zhang, X. et al., Journal of Combinatorial Chemistry, 2006; 8: 705-714.

5. Aurigemma, C. et al., poster presentation, LabAutomation, Palm Springs, CA, USA (2009).

6. Chen, R. and Subbarao, L., unpublished results, 2009.

7. White, C, and Burnett, J., J. Chromatog A., 2005; 1074, 175.

8. Mich, A, Matthes, B, Chen, R, and Buehler, S., LCGC Europe, the Application book, March 2010.