Background
• Analytical SFC methods developed on Chiralpak AY
• Modifiers of methanol, ethanol and isopropanol showed baseline separation of the enantiomers
• Attempt to scale up using pSFC with AY column up to 70% ethanol failed
• Desired enantiomer did not elute
2
Preparative SFC trace using AY column
At 70% ethanol, desired enantiomer did not elute even after 30 minutes.
Switched to 70% methanol (very broad desired enantiomer eluted).
3
Column: AY-H, 30 X 250 mm, 5µ
Isocratic: 70% Ethanol
Flow: 110 mL/min
Loading: 200 mg
Undesired enantiomer
“Catch and Release” like purification technique
• Possibility of “catch and release” like technique using flash chromatography ensued due to retention behavior of enantiomers on AY column and ethanol as the mobile phase
• Due to time constraint and unavailability of a larger preparative AY column, “catch and release” like technique on preparative SFC instrument was not feasible
4
Interactions - “Catch & Release” using SPE
vs. “Catch & Release” using CSP 5
• “Catch and release” is widely used in Ion Exchange
separation with SPE. The sorbent (anion or cation exchange)
traps the compound of interest via electrostatic interaction
and eluted with an acidic or basic modifier
•The “catch and release” technique using CSP may be
hydrophilic and hydrophobic interactions depending on which
sorbent is used
Catch and Release using SPE
Step 1 – Condition SPE with
solvent A
Step 2 – Add sample
Step 3 – Wash with solvent A to
elute unwanted compound
Step 4 – Elute compound of
interest with solvent B
6
Catch & Release using CSP
Step 1 – Condition CSP with
solvent A
Step 2 – Add racemate
Step 3 – Wash with solvent A to
elute enantiomer 1
Step 4 – Wash with solvent B to
elute enantiomer 2
Step by step “Catch & Release” using SPE
vs. “Catch & Release” using CSP
vs
mi0 2 4 6 8 10
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mi0 2 4 6 8 10
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mi0 2 4 6 8 10
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mi0 10 20 30 40 50
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LC method development using
analytical AY-H column 7
IpOHDesired
UndesiredMeOH
Desired
Undesired
EtOH
Desired
Undesired MeCN
Desired
Undesired
MeCN selected to release desired enantiomer based on elution profile and peak shape
Isopropanol (IpOH) and Methanol (MeOH): Almost baseline resolution, desired peak very broad (> 3 minute from baseline to baseline) Acetonitrile (MeCN): Partial resolution between enantiomers. Desired peak eluted in less than 2 minutes (k’ = 1) ( < 2 minutes from baseline to baseline) Ethanol (EtOH): Desired enantiomer eluted 40 minutes after the undesired enantiomer ( = 11.4)
Testing the concept -
Application of LC method to “catch and release”
the enantiomers using Chiral Stationary Phase (CSP)
• 200 gram of 20 µ Chiralpak AY CSP
is slurry packed with ethanol into a
500 mL vacuum vessel
• 5 g of crude racemate is dissolved
in ethanol and manually loaded
onto bed
• Step 1 - CSP (column) washed
with 12 column volumes (7
fractions) of ethanol applying low
vacuum
• Step 2 - Column was washed with
12 column volume (11 fractions) of acetonitrile while applying low vacuum
8
The set up
mi0 1 2 3 4 5 6 7 8 9
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Step 1 – HPLC traces of fractions
collected using ethanol 9
F1F7
• Desired enantiomer was not observed (7 fractions collected)
• Fraction 7 (F7)showed only a trace of the undesired enantiomer
Collected fractions profile
Fraction monitoring conditionsColumn: Chiralpak AY-H 150x4.6 Mobile Phase: MeCN (neat)
Flow Rate: 1.5 mL/min Sample: 5 uL of each fraction collected
mi1 2 3 4 5 6 7 8 9
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QC of fractions collected from step 1 by HPLC 10
min 1 2 3 4 5 6 7 8 9
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mAU
Racemate
Undesired
(pooled F2-F6)
Column: Chiralpak IF, 150x4.6, 5µ
Mobile Phase: 50% CO2(l) / 50%
MeOH containing 20mM Ammonia
Flow Rate: 4 ml/min
BPR: 160 Bar
Column Temp: Ambient
Pooled fractions recovery
• Fraction 1 – ~0.3 g
• (>98% e.e., 93% chemical purity)
• Fractions 2 – 7: ~0.9 g
• (>98% e.e., 97% chemical purity)
• Total recovery F1-F7 = 89%*
*adjusted for the %purity of crude (54%)
* Elution order is reversed using IF column (Column switched to IF because
the analytical AY was being used to monitor the fractions and no back up
column available)
Desired
Undesired
mi2.5 5 7.5 10 12.5 15 17.5
mA
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11
Fractions monitoring conditionsColumn: Chiralpak AY-H 150x4.6 Mobile Phase: MeCN (neat)
Flow Rate: 1.5 mL/min Sample: 5 uL of each fraction collected
Solid Blue Line – F1
Dashed Purple Line – F11
Impurity
Desired
• Undesired enantiomer was not observed (11 fractions collected)
• Fraction 11 showed no trace of the desired enantiomer
Step 2 – HPLC traces of fractions
collected using acetonitrile
Collected fractions profile
12QC of fractions collected from step 2 by HPLC
min 1 2 3 4 5 6 7 8 9
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1400 mAU
Desired
(pooled F5-F9)
Column: Chiralpak IF, 150x4.6, 5uM
Mobile Phase: 50% CO2(l) / 50% MeOH containing 20mM Ammonia
Flow Rate: 4 ml/min
BPR: 160 Bar
Column Temp: Ambient
Pooled fractions recovery• Fractions 1-5 – ~0.9 g
• (>98% e.e., 60% chemical purity)
• Fractions 6–9: ~0.6 g
• (>99% e.e., 97% chemical purity)
• Total recovery F1-F9 = 84%** Adjusted for the %purity of crude (54%)
* Elution order is reversed using IF column
mi1 2 3 4 5 6 7 8 9
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Desired
Undesired