Application Note

Accelerating your process optimization: samplingfrom reactions in-progress means better decisionsin less time
Introduction and background

5–25 mL working volume range. Each reaction
vessel can be independently heated to 150 °C,
cooled to -20 °C and pressurized from ambient to
400 psi above ambient to tightly controlled limits.
The automation allows samples to be taken fromall eight reactors without halting the process —even under high temperatures and pressures, andwith minimal loss of solvent or change of massbalance.

Drug development and fine chemical researchers
are always searching for the best reaction conditions — those that give the greatest yield, the highest purity or the most reaction control, using themost cost effective reagents and conditions. Getting detailed knowledge of how a reaction performs
under a variety of conditions is ideal, but the levelof detail needed to make optimal process decisions
is usually unobtainable due to time and resource
constraints. As a result, your process is limited byinadequate information. Times have changed andtechnology exists that can give you a better understanding of a reaction profile and enable criticalscale-up and control strategies.

Scientists can easily build kinetic reaction profiles, detect any potential impurities and optimize
these reactions with minimal material and timerequirements. All results are funneled to a central
database through the Lab Execution and Analysis(LEA) software suite. LEA, Unchained Labs' enterprise data solution, is an end-to-end solutionthat captures all relevant data from an experimentand stores them in a central database under one
experimental ID. Data that spans from initial reac-The Optimization Sampling Reactor (OSR) fromUnchained Labs is a proven automation tool thatlets researchers study reaction kinetics, track
conversion and impurity formation and determine the reaction end-point over short and longtime scales, all without running extra reactions orhaving to use large amounts of material. You can
get the right data and enough of it, which allows
you to optimize processes faster while increasing scale up success. In this application note, wedemonstrate how the OSR technology was usedin our search for the best chemistry for OSR validation.

The Optimization Sampling
ReactorThe OSR module (Figure 1) is comprised of eight
40 mL overhead stirred reactors, each with a

Figure 1: The Optimization Sampling Reactor (OSR) and controlled access sampling port.

1ACCELERATING PROCESS OPTIMIZATION

1). Both OSR needle
and antechamber gas supply
tip are inserted into the antechamber
2). Seals formed with O-ring
3). Antechamber pressured tobe equal to reactor’s pressure

OSR needle
Antechamber
gas supply tip

Antechamber
Valve betweenantechamberand reactor1). Valve opens2). Needle enters
reactor for
injection/samplingValveremains
closedReactorReactor

ReactorFigure 2: Controlled atmosphere injection and sampling under pressure.

tion design to OSR processing conditions and evenanalytical results (such as HPLC from third partyequipment) can be viewed and analyzed from anywhere for more efficient reporting and seamlessknowledge sharing.

reach the reactor’s pressure. Next, a normally closed
valve is actuated after pressure equilibrium to let theneedle move into the reactor. The stirring paddle for
the OSR (Figure 3) has a hollow shaft, allowing theneedle to travel through without interfering with thestirring mechanism. Dispensing occurs at the top of
the reactor while sampling takes place near the verybottom. This design feature also allows sampling totake place with continuous stirring (slurry sampling)which is ideal for obtaining representative samples
from heterogeneous reactions. Under a different
scenario, the stirring can also stop completely before
sampling, allowing heterogeneous catalysts to settle
so no catalyst is taken out of the reaction. In addition
to time point sampling, the OSR can also monitor
gas uptake in real-time and provide scientists withan additional method to track reaction progress forpressurized reactions.

Accurate sampling anddispensing at elevated pressures
Pressurized chemistry usually provides a technicalchallenge for researchers who want to remove timeseries samples from their reactions to study kinetics and impurity profiles. The compromise is often
to replicate the reaction multiple times and quench
each reaction at a desired time point. This approach is
not only time-consuming, but can also result in morevariability due to inconsistency in setting up replicates.
Accurate, automated in-situ sampling and dispensing of new reagents, even at high temperatures andpressures, is a much better approach. Each condition
is run only once and many sampling and dispensing
points can take place during the run as desired, using
the OSR’s controlled atmosphere sampling port technology (Figure 2).
In order for the OSR needle to inject or sample fromeach reactor under pressure, it has to first enter anantechamber for pressure equilibration. First, theneedle and antechamber gas supply tip are inserted into the antechamber under ambient pressure.
There are O-rings for both to ensure pressure seals
when the needle and gas supply tip enter. The antechamber then is pressurized via the gas supply tip toFigure 3: OSR stirring paddle.

2ACCELERATING PROCESS OPTIMIZATION

Hydrogenation of trans-Cinnamic acid
O2-Methyltetrahydrofuran (2-MeTHF) as thesolvent. The chemistry is not sensitive and both thestarting material and the catalyst can be handled
and weighed in a chemical hood. The conversion
to Hydrocinnamic acid is always clean without
detection of any impurity.

OOH

Catalyst, 2-MeTHF

OH

50 psi of H2, 40 °C
trans-Cinnamic Acid

Sample variation testing

Hydrocinnamic Acid

Prior to running the hydrogenation study, theprecision and accuracy of pressure sampling wastested from 100–1000 μL under 0–400 psi ofpressure using 2-MeTHF as the testing solvent(same solvent as for the hydrogenation reaction).
Sample variation was less than 4% using the OSR(Figure 5).

Figure 4: Hydrogenation of trans-Cinnamic acid.

For every OSR built, a robust and easy-toperform chemistry is needed to evaluate its
performance both in-house and at a customer
site when delivered. Catalyzed hydrogenation oftrans-Cinnamic acid is chosen due to its simplicity(Figure 4). There are only four components
needed for this chemistry: trans-Cinnamic acidas the starting material, H2 gas, catalyst and5% Pd/C
5% Pd/C was first to be evaluated as the catalyst.
The chemistry typically involves 5 mg of 5% Pd/Cin 2-MeTHF at 40 °C under 50 psi of H2.

1200

TargetAverage actual
1000

Volume (µL)

800

600

400

200

0

00

0

0

50

50

50

50

200

200

200

200

200

200

200

200

Sampling pressure (PSI)% accuracy(average actual/target)% RSD (precision)
Target volume(µL)

0psi

50
psi

200
psi

400
psi

0psi

50
psi

200
psi

400
psi

100

2.6%

0.3%

2.6%

3.1%

95%

105%96%

103%

200

0.9%

1.1%

1.6%

1.1%

99%

102%

98%

105%

500

0.5%

0.3%

0.7%

0.9%

99%

101%

98%

102%

1000

0.2%

0.1%

0.6%

0.1%

100%

102%

100%

100%

Figure 5: Sampling precision and accuracy at pressure.

3ACCELERATING PROCESS OPTIMIZATION

12

3

4

5

6Figure 6: The freeslate jr. deck layout. (1) solvent tray, (2) OSR, (3) 3-position passive rack, (4) wash station, (5) heating/cooling/stirring
station and (6) heating/stirring station.

Run Design:

5 mg of Pd catalyst was first added as powders toeach OSR vial. It was followed by transferring 20mL 50 mg/mL solution of trans-Cinnamic acid in2-MeTHF to each vial. The OSR was sealed, andthe heterogeneous solution stirred for approximately 10 minutes at room temperature while
purging with nitrogen. A t=0, 100 μL sample wastaken from each OSR reactor.

• Start material: 20 mL 50 mg/mL trans-Cinnamic
acid in 2-MeTHF solution• Catalyst: 5 mg 5% Pd/C
• Process gas: H2 at 50 psi• Paddle speed: 500 rpm• Sampling intervals (min): 0, 24, 48, 72, 96

2 Add 20 mL 50 mg/mL trans-Cinnamic acidsolution to each reactor vial

Once t=0 sampling was complete, the reactors
were heated to 40 °C and purged with 3 x 50 psiof H2 to replace existing N2. The reactors werepressurized one more time with 50 psi of H2 tocommence the reactions. Samples (100 μL) werethen taken from each reactor at 24, 48, 72 and 96
minutes. Once taken, samples were transferred toa microtiter plate located on the freeslate jr. deck(Figure 6) to be analyzed later by HPLC.

3 Seal and purge the OSR with N2Results

4 Start stirring at 500 rpm

The chemistry was done in about an hour. The
percent conversion over time was then calculated
based on HPLC data from each time point sampled(Figure 7). Standard deviation is less than 5% for alltime points sampled.

• Analytics: HPLC after 50x dilution of samples with
30/70 THF/MeCN

Procedure:
1 Pre-weigh 5 mg 5% Pd/C and add to each reactor vial

5 Heat OSR to 40 °C, followed by 3 x 50 psi of H2
purge to replace N2
6 Take time series samples (100 µL) at statedintervals
7 Analyze HPLC after 50x dilution of samples with
30/70 THF/MeCN% Conversion =4

Product area (Hydrocinnamic acid)
Total area (trans-Cinnamic acid + Hydrocinnamic acid)100%

100%90%90%80%80%70%70%% Conversion% Conversion (product area/total area)

ACCELERATING PROCESS OPTIMIZATION

Reactor A
Reactor B
Reactor C
Reactor D
Reactor E
Reactor F
Reactor G
Reactor H

60%50%40%30%50%40%30%20%20%10%10%0%020

406080100

Reactor A
Reactor B
Reactor C
Reactor D
Reactor E
Reactor F
Reactor G
Reactor H

60%0%120020406080120100Time points (mins)

Sampling time points (mins)

Figure 7: Percent conversion for 5% Pd Run based on HPLC data.

Figure 8: Percent conversion for 5% Rh on alumina run (two different supports) based on HPLC data..

This Pd chemistry has been working well for us
and we have been using it as part of our OSR
development for a long time. There was just one
noticeable issue. Depending on the locations,
5 mg 5% Pd/C can be difficult to accurately
weigh due to static. Since overall catalystamount is small, any inaccurate weighing would
directly impact consistency in activity between
reactors. As stated earlier, the ultimate goal
was to find a very robust procedure that would
reliably evaluate OSR performance with minimal
difficulties conducting it in-house and at acustomer site.

212857) and the Degussa type support (Aldrich663468) for 5% Rh on Alumina, were selected togauge their effects on reaction rates. The study design includes four replicates for each support type(50 mg each) to assess consistency across different
reactors (Table 1). The rest of the run design andprocedure are the same as the 5% Pd run.
Reactions catalyzed by matrix-supported Rh (Reactor A, C, E and G) are clearly more active thanDugussa type (Reactor B, D, F and H) (Figure 8).
All four replicates are also very consistent and theCV for all time points was <1% across eight reactors
for all 48 time points.

5% Rh on alumina on two different
supports

Degussa Type 5% Rh on alumina
Slower Dugussa type catalyst is ideal for validation
chemistry since it allows more evaluation (time)
points before all reactions hit 100% conversion.
The catalyst is also easy to weigh and has no static issues.

One idea was to use less active catalysts so morecatalysts are needed and variability in weighing
becomes less of a factor. 5% Rh on alumina waschosen due to its known low activity. Two different catalyst supports, the matrix support (AldrichReactor

AMatrix support (mg)50

Degussa support (mg)

BC

D50E

F

505050

Table 1: Catalyst loading for 5% Rh on alumina run involving two different supports.

5GH505050

ACCELERATING PROCESS OPTIMIZATIONreactions that successfully optimized a pressurizedchemistry. For each series, a total of 40 (5 pointsx 8 reactors) time points were taken automatically in-situ and under pressure in two hours, wheretraditionally each individual time point must be setup as a reaction. The OSR’s ability to gather significant amounts of data demonstrates how its use
can contribute to a much greater understandingof reaction kinetics over a short period of time. In
addition, the OSR’s controlled sampling port technology allows experiments to be run in semi-batchmode where materials are added and taken away
at pressure, which aligns with many plant-based
operations where the addition of new reagents andsampling from the vessel can take place withouthalting the process.

100%90%80%% Conversion

70%Reactor A
Reactor B
Reactor C
Reactor D
Reactor E
Reactor F
Reactor G
Reactor H

60%
50%
40%30%20%10%0%020

40

60

80

100120Time points (mins)

Figure 9: Percent conversion for 5% Rh on alumina run (Degussasupport) based on HPLC data.

Unchained Labs’ reaction screening and catalysis
optimization workflows for pharmaceutical process
research, including OSR technology, allow scientists to increase the number of experiments they
can perform while also improving the fidelity andtraceability of their data. The workflows enabled
by freeslate or freeslate jr. are compatible with awide range of reactions and backed by Unchained
Labs' many years of industry experience anddomain knowledge. Scientists can comprehensively view and analyze all relevant data from initialdesign to analytics linked by a single experimental ID immediately or years from now. With thesesolutions, researchers can now get the right dataand plenty of it in much less time, empoweringdevelopment teams to make more informed decisions as processes are transferred and increase
the chance for success at scale-up.

Validation chemistry based on Dugussa-support
5% Rh on alumina (50 mg) was then evaluated(Figure 9). Eight replicates were complete in about
an hour (same as prior run) with great consistenty(%RSD is less than 5% for all time point samples).
Using 50 mg Degussa type 5% Rh on alumina to catalyze hydrogenation of trans-Cinnamic acid reaction
is currently our standard validation chemistry.

Conclusion
This study first demonstrated OSR’s sampling
capabilities at low volumes (100 μL) and under highpressures (up to 400 psi) with sample variation atless than 4%. It was followed by three quick series ofUnchained Labs
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