U.S. patent application number 09/728751 was filed with the patent office on 2002-01-17 for method for screening multiple reactants and catalyst systems using incremental flow reactor methodology.
Invention is credited to Flanagan, William Patrick, Sabourin, Cheryl Lynn, Spivack, James Lawrence.
Application Number | 20020007093 09/728751 |
Document ID | / |
Family ID | 26830998 |
Filed Date | 2002-01-17 |
United States Patent
Application |
20020007093 |
Kind Code |
A1 |
Flanagan, William Patrick ;
et al. |
January 17, 2002 |
Method for screening multiple reactants and catalyst systems using
incremental flow reactor methodology
Abstract
Method for producing multiple chemical reactions and for rapid
screening of chemicals, catalysts, process conditions and the like
is disclosed. The method includes the steps of providing an array
of reactor vessels and reactants; loading each reactor vessel with
at least one reactant; and allowing the reactions to proceed for a
predetermined time interval. A volume increment is withdrawn from
each reactor vessel and a volume increment of at least one reactant
is added to each reactor vessel in the array. The steps of volume
increment withdrawal and addition are repeated after successive
time intervals until the reactions reach a substantially steady
state. The loading, withdrawal, and addition steps are performed by
liquid or solid handling robots. In one embodiment, the volume
increment withdrawal occurs before, after, or contemporaneously
with the volume increment addition.
Inventors: |
Flanagan, William Patrick;
(Rexford, NY) ; Spivack, James Lawrence;
(Cobleskill, NY) ; Sabourin, Cheryl Lynn;
(Schenectady, NY) |
Correspondence
Address: |
General Electric Company
CRD Patent Docket Rm 4A59
P.O. Box 8, Bldg.K-1 - Salamone
Schenectady
NY
12301
US
|
Family ID: |
26830998 |
Appl. No.: |
09/728751 |
Filed: |
December 4, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09728751 |
Dec 4, 2000 |
|
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09443640 |
Nov 18, 1999 |
|
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60133061 |
May 7, 1999 |
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Current U.S.
Class: |
568/723 |
Current CPC
Class: |
B01J 2219/00691
20130101; B01J 2219/00373 20130101; B01J 2219/0072 20130101; B01J
19/0046 20130101; B01J 2219/00367 20130101; C40B 60/14 20130101;
B01J 2219/0068 20130101; B01J 2219/00283 20130101; B01J 2219/00335
20130101; B01J 2219/00414 20130101; B01J 2219/00596 20130101; B01J
2219/00481 20130101; B01J 2219/00497 20130101; B01J 19/0006
20130101; B01J 4/02 20130101 |
Class at
Publication: |
568/723 |
International
Class: |
C07C 039/16 |
Claims
What is claimed is:
1. A method for producing multiple chemical reactions and catalytic
systems comprising the steps of: (a) providing an array of reactor
vessels and reactants; (b) loading each of the reactor vessels with
at least one reactant; (c) allowing the reactions to proceed for a
predetermined time interval; (d) withdrawing a volume increment
from each reactor vessel; (e) adding a volume increment of at least
one reactant to each reactor vessel; and thereafter (f) repeating
steps (c), (d), and (e) until such time the reactions reach a
substantially steady state.
2. The method of claim 1, wherein the reactants include phenol and
acetone.
3. The method of claim 1, wherein the reactions result in the
formation of bisphenol A.
4. The method of claim 1, wherein the volume increment comprises
phenol and acetone.
5. The method of claim 2, wherein the reactants further include an
acid catalyst.
6. The method of claim 2, wherein the reactants further include a
reaction promoter.
7. The method of claim 1, wherein the volume increment withdrawal
occurs before, after, or contemporaneously with the volume
increment addition.
8. The method of claim 1, wherein the loading, withdrawal, and
addition steps are performed by a liquid or solid handling
robot.
9. The method of claim 1, further including the step of controlling
the size of the volume increments withdrawn and added and the time
interval between the additions of the volume increments to obtain a
desired reactor residence time.
10. The method of claim 1, wherein the volume increments are
withdrawn from the reactor vessels by positioning a probe at a
predetermined level in the reactor vessels and withdrawing reactor
fluid until no further fluid can be withdrawn at that level .
11. The method of claim 1, wherein the time intervals and the
volume increments are selected to obtain a desired space velocity
defined by the following equation: wherein: .DELTA.t time interval
[min]; .DELTA.V=volume increment [.mu.L]; .rho.=density of the
volume increment added [g/mL]; SV=space velocity [g liquid feed/g
resin/hr]; and R=amount of resin [mg].
12. The method of claim 1, wherein the withdrawal and addition
steps are controlled in the reactor vessels so as to produce
sub-interval concentration gradients during the course of the
reactions.
13. The method of claim 12, wherein large volume additions followed
by sequential withdrawals of smaller volume increments are made to
the reactor vessels at predetermined subintervals within the time
interval.
14. The method of claim 1, wherein the volume increments withdrawn
from the reactor vessels are analyzed for properties of
interest.
15. The method of claim 1, wherein the volume increments withdrawn
from the reactor vessels are pooled and then analyzed to provide
cumulative data.
16. The method of claim 1, wherein after the reactions are allowed
to proceed for a predetermined time interval, the volume increments
are withdrawn and added simultaneously in each of the reactor
vessels.
17. A method for high throughput screening of chemicals, catalysts,
reactants, process conditions and the like comprising the steps of:
(a) providing an array of reactor vessels and reactants; (b)
loading each of the reactor vessels with at least one reactant; (c)
allowing the reactions to proceed for a predetermined time
interval; (d) withdrawing a volume increment from each reactor
vessel; (e) adding a volume increment of at least one reactant to
each reactor vessel; and thereafter (f) repeating steps (c), (d),
and (e) until such time the reactions reach a substantially steady
state.
18. The method of claim 17, further including the step of selecting
the predetermined time intervals and the volume increments to
obtain a desired space velocity.
19. The method of claim 18, wherein the desired space velocity is
defined by the following equation: wherein: .DELTA.t=time interval
[min]; .DELTA.V=volume increment [.mu.L]; .rho.=density of the
volume increment added [g/mL]; SV=space velocity [g liquid feed/g
resin/hr]; and R=amount of resin [mg].
20. The method of claim 17, wherein the reactants include phenol
and acetone
21. The method of claim 17, wherein the reactions result in the
formation of bisphenol A.
22. The method of claim 17, wherein the volume increment comprises
phenol and acetone.
23. The method of claim 17, wherein the reactants further include
an acid catalyst.
24. The method of claim 17, wherein the reactants further include a
reaction promoter.
25. The method of claim 17, wherein the volume increment withdrawal
occurs before, after, or contemporaneously with the volume
increment addition.
26. The method of claim 17, wherein the loading, withdrawal, and
addition steps are performed by a liquid or solid handling
robot.
27. The method of claim 17, further including the step of
controlling the size of the volume increments withdrawn and added
and the time interval between the additions of the volume
increments to obtain a desired reactor residence time.
28. The method of claim 17, wherein the volume increments are
withdrawn from the reactor vessels by positioning a probe at a
predetermined level in the reactor vessels and withdrawing reactor
fluid until no further fluid can be withdrawn at that level .
29. The method of claim 17, wherein the withdrawal and addition
steps are controlled in the reactor vessels so as to produce
sub-interval concentration gradients during the course of the
reactions.
30. The method of claim 29, wherein large volume additions followed
by sequential withdrawals of smaller volume increments are made to
the reactor vessels at predetermined subintervals within the time
interval.
31. The method of claim 17, wherein the volume increments withdrawn
from the reactor vessels are analyzed for properties of
interest.
32. The method of claim 17, wherein the volume increments withdrawn
from the reactor vessels are pooled and then analyzed to provide
cumulative data.
33. The method of claim 17, wherein after the reactions are allowed
to proceed for a predetermined time interval, the volume increments
are withdrawn and added simultaneously in each of the reactor
vessels.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of the
filing date of U.S. Provisional Application Ser. No. 60/133,061,
filed May 7, 1999, entitled "INCREMENTAL FLOW REACTOR AND METHOD
FOR PARALLEL SCREENING" and U.S. Non-Provisional Application Ser.
No. 09/443,640, filed Nov. 18, 1999 entitled "METHOD FOR HIGH
THROUGHPUT CHEMICAL SCREENING."
BACKGROUND OF THE INVENTION
[0002] The present invention is generally directed to a method for
the rapid screening of chemicals, catalysts, reactants, process
conditions and the like. More specifically, the present invention
is directed to the use of Incremental Flow Reactor (IFR)
methodology on large arrays of miniaturized reactor vessels to
identify potential reactants and catalyst systems for the bulk
chemical industry.
[0003] Combinatorial chemistry is a popular research tool among
scientists in many different fields. High throughput and
combinatorial screening for biological activity have been prevalent
in the pharmaceutical industry for nearly twenty years. More
recently, high throughput and combinatorial screening for improved
catalysts for the bulk chemical industries have enjoyed increasing
popularity. Despite their popularity, development of high
throughput and combinatorial screening for production scale
reactions has been lagging. This has been due in large part to the
difficulty in emulating the production-scale reactions at the
micro-scale level, which is necessary for this type of work. In
particular, special problems can arise in reactions that are
significantly dependent on flow rate or configuration.
[0004] To date, most combinatorial work has focused on "solid
phase" reactions. It is known that a wide variety of organic
reactions can be carried out on substrates immobilized on resins.
However, a substantial number of production scale reactions are
"liquid phase" or "mixed phase" and are carried out in continuous
flow reactor systems.
[0005] Early efforts in high throughput screening of solutions have
focused on catalyst screening. Before the application of the high
throughput and combinatorial approaches, catalyst testing was
traditionally accomplished in bench scale or larger pilot plants in
which the feed to a continuous flow reactor was contacted with a
catalyst under near steady state reaction conditions. However,
rapid and combinatorial screening of reactants, catalysts, and
associated process conditions requires that a large number of
reactions or catalytic systems be tested simultaneously. In certain
applications, screening-level data can be generated by using
miniaturized batch reactors in conjunction with liquid-handling
robots that aliquot the appropriate catalysts and reactants to each
vial or reaction well. In other applications, however, batch
reactions do not behave in the same fashion as continuous flow
reactions, and could provide misleading results if the goal of
screening is to identify reactants or catalyst systems that will be
implemented in production-scale continuous flow reactors.
[0006] As the demand for bulk chemicals continues to grow, new and
improved methods of producing more product with existing resources
are needed to supply the marketplace. Unfortunately, the
identification of additional effective reactants and catalyst
systems for these processes continues to elude industry. There,
thus, remains a need for new and improved methods for rapidly
screening potential reactants, catalysts, and associated process
conditions.
SUMMARY OF THE INVENTION
[0007] The present invention is directed to the use of IFR
methodology on large arrays of miniaturized reactor vessels to
produce chemical reactions that emulate those carried out in
production-scale, continuous flow or continuous stirred tank
reactors. With IFR, high throughput combinatorial screening of
chemicals, catalysts, reactants, and associated process conditions
is achieved. The use of liquid and solid handling robotic equipment
to implement the IFR on numerous reactor arrays is also
described.
[0008] In one embodiment of the present invention, the method
includes the steps of providing a large array of reactor vessels
and reactants; loading each reactor vessel with at least one
reactant; and allowing the reactions to proceed for a predetermined
time interval. A volume increment is withdrawn from each of the
reactor vessels and a volume increment of at least one reactant is
added to each reactor vessel in the array. The steps of volume
increment withdrawal and addition are repeated after successive
time intervals until the reactions reach a substantially steady
state.
[0009] In an alternative embodiment, the volume increment
withdrawal can take place before, after, or contemporaneously with
the volume increment addition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a graphical representation of concentration
gradients of various reactions;
[0011] FIG. 2 illustrates the IFR method as applied to a single
reaction vial;
[0012] FIG. 3 is a graphical representation of the relationship
among various reaction conditions;
[0013] FIG. 4 is a graphical representation of a reaction kinetics
model comparing a continuous stirred tank reactor with an
incremental flow reactor;
[0014] FIG. 5 illustrates the IFR method as applied to 8 reaction
vials using an 8-probe liquid handling robot; and
[0015] FIG. 6 illustrates the IFR method as applied to a 96-well
micro-titre plate using an 8-probe liquid handling robot.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention is directed to the use of IFR on large
arrays of miniature reactor vessels for the rapid combinatorial
screening of chemicals, catalysts, reactants, and associated
process conditions. Rapid combinatorial screening requires that a
large number of reactions or catalyst systems be tested in
parallel. The method of the present invention produces chemical
reactions that emulate those carried out in production-scale,
continuous flow or continuous stirred tank reactors, and provides
useful information that may be dependent on flow rate and
configuration (e.g., reaction yield; selectivity; and other
reaction characteristics or process variables). With liquid and
solid handling robotic equipment, increments of liquid or solid
flow are delivered to and removed from the arrays of reactor
vessels at predetermined time intervals to mimic the continuous
flow of reactor influents and effluents. The method is particularly
useful for studying the formation of bisphenol A from phenol and
acetone.
[0017] In accordance with the method of the present invention, the
steps comprise
[0018] (a) providing an array of reactor vessels and reactants;
[0019] (b) loading each of the reactor vessels with at least one
reactant;
[0020] (c) allowing the reactions to proceed for a predetermined
time interval;
[0021] (d) withdrawing a volume increment from each reactor
vessel;
[0022] (e) adding a volume increment of at least one reactant to
each reactor vessel; and thereafter
[0023] (f) repeating steps (c), (d), and (e) until such time the
reactions reach a substantially steady state.
[0024] As used herein, the term "substantially steady state" refers
to a point where the reaction effectively emulates a reaction of
interest, such as those carried out in production-scale, continuous
flow or continuous stirred tank reactors. As noted, certain
reaction data are dependent on flow rate, residence time, or
similar parameters. Utilizing the present method, these parameters
can be manipulated in order to obtain useful data on a micro
scale.
[0025] The volume increment withdrawal can take place before,
after, or contemporaneously with the volume increment addition. The
preferred order will depend on the discrete circumstances of a
given application. For example, when working with micro amounts, it
may be preferable to add a volume increment before withdrawal in
order to maintain favorable reaction conditions within the reaction
vessel. In an embodiment, the time increments are selected such
that the withdrawals are made before the reactants present in the
reactor vessels have had a chance to completely react, thereby
ensuring substantially continuous reactivity within the reactor
vessel.
[0026] Each volume increment that is added contains at least one of
the reactants. The term "reactant" means any substance that affects
the reaction in any capacity, including catalysts, promoters, and
the like. The relative amounts of each reactant in the volume
increments can be determined based on the differential depletion,
exhaustion, or inactivation of each species during the course of
the reaction. It is also contemplated that multiple additions of
various reactants and reactant combinations can be made. In one
embodiment, the total volume of the multiple additions is
equivalent to the volume increment withdrawn.
[0027] Volume increments that are withdrawn can be handled in a
number of ways. For example, each volume increment withdrawn from
the reactor vessel can be analyzed individually for properties of
interest. Selected volume increments can be analyzed, while the
non-analyzed volume increments are discarded. Alternatively,
withdrawn volume increments can be pooled to provide cumulative
data for the entire course of the reaction or for selected time
periods of interest.
[0028] In further embodiments of the present invention, automated
liquid or solid robotic equipment is used to deliver and remove the
volume increments from a large array of reactor vessels. Desired
space velocity and reactor residence times can be obtained by
controlling the size of the volume increments withdrawn and added
and the size of the time intervals between volume increment
additions. Unless otherwise noted, time intervals denote the period
of time between successive volume additions.
[0029] The effective liquid residence time in the reactor can be
defined by the following relationship: 1 RT = V tot t V
[0030] wherein:
[0031] .DELTA.t=time interval;
[0032] .DELTA.V=volume increment;
[0033] RT=residence time; and
[0034] V.sub.tot=total liquid volume in the reaction vessel.
[0035] Similarly, the effective liquid flow rate (Q) can be defined
by the following relationship: 2 Q = V t
[0036] It is evident that the behavior of the present incremental
flow method approaches that of a continuous stirred tank reactor as
the time interval and volume increments approach zero: 3 lim t , V
0 ( IFR ) = continuous stirred tank reactor ( CSTR )
[0037] Conversely, as the volume increment approaches the total
liquid volume in the reactor vessel, the behavior of the
incremental flow method approaches that of a sequential series of
batch reactions: 4 lim V V ( IFR ) = sequential series of batch
reactions
[0038] The selection of optimal .DELTA.t and .DELTA.V values will
depend on several factors, including reaction kinetics and the
capabilities of the liquid-handling equipment. As shown in FIG. 1,
a faster reaction will generally exhibit larger concentration
gradients within a given time interval than a slower reaction.
Preferably, for a given reaction system, the .DELTA.t and .DELTA.V
values should be chosen to minimize the within-increment
concentration gradients without placing excessive demands on liquid
handling equipment.
[0039] Accordingly, it may be useful to determine the sub-interval
concentration gradients at various points throughout the course of
the reactions. Not only can this information be useful in verifying
that appropriate .DELTA.t and .DELTA.V values have been chosen, it
could also provide valuable insight regarding reaction kinetics.
Such information can be obtained by establishing reactions of the
type as described above. In other words, allow the reaction to
proceed for a time interval, followed by controlled addition and
withdrawal of nominal volume increments until the reaction reaches
a point of interest. When concentration gradient information is
desired, a sample volume increment is added that is larger (e.g.,
about two to three times larger) than the nominal .DELTA.V. Volume
sub-increments are then withdrawn at appropriate subintervals
within the time interval, such that the sum of the volume
sub-increments is equivalent to the sample volume increment.
Analysis of the withdrawn sub-increments provides desired
concentration gradient data. The reactions are allowed to continue
until sub-interval concentration gradient information is again
desired, at which point the steps for obtaining such information
can be repeated.
[0040] In a further alternative embodiment, volume increment
withdrawals are effected by inserting a probe to a predetermined
level in the reactor vessels and withdrawing reactor fluid until no
further fluid can be withdrawn. In this manner, the probe acts as a
liquid level controller, thereby ensuring that the liquid level in
the reactor vessels will be the same at the end of each time
interval. When using a robotic probe, the efficacy of this approach
depends, inter alia, on how accurately and reproducibly the probe
can be positioned at the desired liquid level.
[0041] This embodiment reduces or eliminates the possibility of
cumulative volume error related to the accuracy of incremental
volume withdrawals and also compensates for error related to the
accuracy of incremental volume additions. For example, if a
slightly larger than desired volume increment is added at the
beginning of a time interval, a similarly larger volume increment
will be withdrawn at the end of that time interval since the volume
increment removal is based on a liquid level control mechanism.
Conversely, a smaller than desired volume increment addition would
be compensated for by a smaller volume increment removal.
EXAMPLES
[0042] The following prophetic example is based on a mathematical
reaction kinetic model and compares incremental flow reactor
behavior with continuous stirred tank reactor behavior.
[0043] The dihydric phenol 2,2-bis(p-hydroxyphenyl)propane
(commonly referred to as "bisphenol-A", "BPA" or "pp-BPA") is
commercially prepared by condensing 2 moles of phenol with a mole
of acetone in the presence of an acid catalyst. The phenol is
typically provided in molar excess of the stoichiometric
requirement. Optional reaction promoters, such as free mercaptans,
can be added to aid the reaction. Common acid catalysts for the
production of BPA include acidic ion exchange resins, such as
sulfonic acid, substituted polystyrene, and the like.
[0044] For purposes of discussion, assume that the ion exchange
resin-catalyzed formation of BPA from phenol and acetone is
conducted in continuous-flow reactors at a space velocity of 2.33 g
liquid feed/g resin/hr. For a small vial containing 150 mg resin
and 1000 .mu.L liquid volume, the corresponding liquid flow rate
for a true continuous flow reactor would be 338 .mu.L/hr (assuming
a liquid feed specific gravity of 1.018 g/mL). To mimic continuous
flow in the small vial example using the IFR method, the following
sequence would be followed.
[0045] Referring to FIG. 2, each vial or reaction well is loaded
with the appropriate mixture of phenol:acetone feed 12. The feed
can contain optional promoter(s) and catalyst(s). Each vial is
provided with resin beads 16 and an optional stir bar 18.
[0046] The reaction is allowed to proceed in batch mode for one
time interval, .DELTA.t. Near the end of this time interval, a
probe (not shown) withdraws one liquid volume increment, .DELTA.V,
of reaction mixture 14 from the vial (reactor effluent). The
withdrawn volume increment is replaced with an equal volume
increment, .DELTA.V, of fresh feed 12. Cycle time, .DELTA.t, is
defined as the time period between successive volume increment
additions. The incremental withdrawal and addition of reactants is
continued until the reaction reaches a substantially steady state,
and screening data are collected.
[0047] The values of the time intervals and volume increments
(.DELTA.t and .DELTA.V) can be selected to obtain a desired space
velocity. The relationship between the time intervals and volume
increments is as follows:
[0048] wherein:
[0049] .DELTA.t=time interval;
[0050] .DELTA.V =volume increment;
[0051] .rho.=density of liquid feed;
[0052] SV=space velocity; and
[0053] R=amount of resin.
[0054] The relationship between .DELTA.t and .DELTA.V is
illustrated in FIG. 3 for the present example (V.sub.tot=1000
.mu.L; resin amount=150 mg/L; space velocity=2.33 g liquid flow/g
resin/hr), along with results for a range of other space
velocities.
[0055] FIG. 4 is a comparison of the IFR method and a traditional
continuous stirred tank reactor (CSTR). The plots were generated
using mathematical reaction kinetics models with the following
parameters:
1 .DELTA.t = 5.7 minutes k.sub.pp = 1 hr.sup.-1 .DELTA.V = 30 .mu.L
k.sub.op = 0.005 hr.sup.-1 Space velocity = 2.33 g liquid feed/g
resin/hr k.sub.isofwd = 0.01 hr.sup.-1 Resin amount = 150 mg
k.sub.isorev = 0.01 hr.sup.-1 Liquid volume in reactor = 1000 .mu.L
k.sub.alk = 0.01 hr.sup.-1 Initial acetone in reaction vials = 0 wt
% Acetone in feed = 4.67 wt %
[0056] The hypothetical model reactions are shown below: 1
[0057] It should be apparent from the graphic representations in
FIG. 4 that the IFR method closely emulates the CSTR under the
stated conditions.
[0058] To enable high-throughput combinatorial screening of
chemicals, catalysts, and process conditions, the IFR method was
used on many arrays of miniaturized reactor vessels using liquid
handling robotic equipment such as the Gilson Multiprobe 215 Liquid
Handler (Middleton, Wis.). Experimental data was generated using
the IFR methodology on two systems: 1) a single-probe liquid
handling robot to operate a one-dimensional array of 12 IFRs (1
column.times.12 rows); and 2) an 8-probe liquid-handling robot to
operate a two-dimensional array of 96 IFRs (8 columns.times.12
rows).
[0059] The following modifications were made to the Gilson
Multiprobe 215 Liquid Handler. These modifications were necessary
in order to work with phenol or other chemicals that are solid
(rather than liquid) at room temperature. It should be noted that
if the chemicals being used are liquid at room temperature, the
modifications described below would not be necessary to implement
the IFR methodology.
[0060] Heat-traced all transfer lines. The transfer lines are
flexible tubes connecting the individual syringe pump heads to the
liquid-handling probes. These lines contain a "system fluid." In
order to collect a liquid sample with the liquid-handling probe, a
motor-driven syringe pulls a desired volume of system fluid through
the transfer line which, therefore, draws the same volume of sample
fluid into the probe. To dispense the sample fluid from the probe,
the motor-driven syringe pushes the desired volume of system fluid
out through the transfer line, thereby displacing the sample fluid
from the probe. The transfer lines must be kept warn (60.degree.
C.) in order to prevent "freezing" of the sample fluids in the
probe. The transfer lines are, therefore, sheathed in an
electrically-heated wrapping.
[0061] Heat-traced rinse station to prevent freezing of
phenol-containing rinsates. After collecting and dispensing a
particular liquid sample, the probes can be rinsed with system
fluid by lowering the 8 probes into a rinse station and flushing
with system fluid. The system fluid is pumped out through the
probes and then flushed out of the rinse station to a drain line.
The rinse station is electrically heated to prevent freezing of
phenol-containing rinsates.
[0062] Heat-traced drain line to prevent freezing of
phenol-containing rinsates in the line leading from the rinse
station to the waste collection reservoir. In the present
invention, 1/4" copper tubing was used for the drain line. The
tubing was wrapped with commercially-available heat tape
(electrical) to keep it warm.
[0063] Several heating blocks were custom-built to keep the
chemicals warm during the experiments. The heating blocks were
mounted on the liquid-handling robot's deck. Each is described
individually as follows:
[0064] a) Heating block for stock solutions. Stock solutions
(containing reactants such as phenol, acetone, and promoter) were
stored in 48-well deep-well micro-titre plates. These solutions
were the "feeds" to the 96 reaction vials. The 48-well plates were
clamped within an aluminum frame and bolted to an aluminum base.
The base was heated with electrical cartridge heaters. Power to the
electrical heaters was regulated by a temperature controller based
on feedback from a thermocouple mounted in the aluminum base.
[0065] b) Heating block for phenol reservoirs. Phenol was used for
rinsing of the probes, and for insulating the feeds and samples
(i.e., the "incremental volumes") from the system fluid. In other
words, before the probes collected feed or sample volumes, they
first collected a small volume of fresh phenol, then a small air
gap. This was done so that the feed and sample volumes did not
become contaminated by direct contact with the system fluid. The
fresh phenol used for this purpose had to be kept warm, so
glass-lined or polypropylene-lined aluminum "boxes" were
constructed to hold the molten phenol. The boxes were mounted on an
aluminum base that was electrically heated as previously described.
In this case, the power to the electrical heaters was regulated by
a temperature controller based on feedback from a thermocouple
placed in the Al wall of the boxes.
[0066] c) Heating block for 96-well reactor array. The reactor
wells or vials must also be kept warm since they contain phenol. In
the present invention, a form-fitting aluminum mold was fabricated
to fit into the underside of the 96-well polypropylene micro-titre
plate that was used as the reactor array. The assembly was placed
into a Reacti-Therm III Heating/Stirring Module (Pierce; Rockford,
Ill.), which kept the reactor array assembly at the desired
temperature. This modification is required for the IFR (independent
of whether one uses a high melting solvent or not) because reaction
temperature control is essential: the Al mold reduced well to well
T variation from about 2-3.degree. C. to about 0.2.degree. C.
[0067] The choice of system fluid is quite important. Molten phenol
(with an additive to help keep it from freezing) was initially used
as a way to minimize contamination of the feed and sample volumes
with foreign chemicals in the system fluid. It was discovered,
however, that the phenol was swelling the internal parts of the
valves in the syringe pump assembly, thereby resulting in valve
failure. To avoid this problem, a suitable solvent is now used as
the system fluid. Contamination of feed and sample volumes is
prevented by the aforementioned use of a phenol/air gap between the
system fluid and the feed or sample volumes.
[0068] Various embodiments of the present invention are described
below.
[0069] A single-probe Gilson 215 liquid-handling robot was used to
implement the IFR methodology on a limited number of reactor
vessels, for example, a one-dimensional array of 12 IFRs. The
robotic probe sequentially addressed each reactor in the array
until the entire array was addressed. The robotic probe then
returned to the first reactor in the array and repeated the
process. The IFR methodology was implemented in several different
ways.
[0070] The robotic probe removed a liquid volume increment from the
first reactor, and then immediately delivered an increment of fresh
feed to the first reactor. This process was then repeated for the
second reactor, then the third, and so on, until all reactors in
the array had been addressed. The robotic probe then returned to
the first reactor and repeated the process. In this manner, a
single "time interval" of the IFR method was carried out each time
the robotic probe cycled through the array of reactor vessels.
[0071] The robotic probe removed a liquid volume increment from the
first reactor, then removed a liquid volume increment from the
second reactor, and so on, until liquid volume increments had been
removed from all reactors in the array. Then, the robotic probe
delivered an increment of fresh feed to the first reactor, then
delivered an increment of fresh feed to the second reactor, and so
on, until all reactors in the array had been addressed. In this
manner, the robotic probe cycled through the array of reactor
vessels twice in order to carry out a single "time interval" of the
IFR method.
[0072] Other variations of the IFR methodology can also be
implemented as described in U.S. patent application Ser. No.
09/443,640, the reference being hereby incorporated by reference.
For example, large volume additions followed by sequential removals
of smaller volume increments can be used to obtain reaction kinetic
data at various points throughout the course of the reaction.
Alternatively, two additions of different reactants can be followed
by removal of single or multiple volume increments. Further, single
or multiple additions of multiple reactants can be followed by
removal of single or multiple volume increments.
[0073] The single-probe approach is not limited to the specific
examples described herein. The IFR method can be applied to either
one-dimensional or small two-dimensional arrays. In practice,
however, the number of reactors that can be addressed with a
single-probe robot is limited by the ability of the robot to
deliver and remove liquid volume increments to all the reactors in
the array at the desired time intervals.
[0074] An eight-probe liquid-handling robot was used to
conveniently implement the IFR methodology on a 96-reactor
(8.times.12) array. In the method of the present invention, the
eight robotic probes aligned directly with the first row of eight
reactors as shown in FIG. 5. The eight probes simultaneously
addressed the eight reactors in the first row, and then moved on to
the second row, and so on, until all rows in the array had been
addressed. Any variation of the IFR methodology discussed above can
also be implemented using the eight-probe liquid-handling
robot.
[0075] Any type of liquid-handling robot which is fitted with any
number of probes or tips can also be used to implement the IFR
methodology on an array of reactor vessels. Further, any robotic
liquid-handling device which utilizes an array of probes or pipette
tips that is geometrically identical to the array of miniature
reactor vessels can be used to simultaneously implement the IFR
methodology on all of the reactor vessels in the array. For
example, a commercially-available robotic liquid handling device
equipped with 96 tips in a standard 8.times.12 array can be used to
simultaneously address a 96-reactor array as shown in FIG. 6.
Liquid volume increments can be simultaneously removed from all 96
reactors, and then liquid volume increments of fresh feed is
simultaneously added to all 96 reactors.
[0076] The IFR methodology may be applied to any type or geometric
configuration of miniature reactor arrays. This includes: 1)
micro-titre plates of any size, including 48 wells (8.times.6
array), 96 wells (8.times.12 array), 384 wells (15.times.24 array),
1536 wells (32.times.48 array), or any other number of wells; and
2) any array of glass (or metal or plastic or any other material)
vials, tubes, bottles, cups, or any other suitable container. FIG.
6 illustrated the IFR method as applied to a 96-well micro-titre
plate using an 8-probe liquid-handling robot. The robot
simultaneously addresses 8 reaction wells in a single row, then
moves to the next row, etc. The micro-titre plate has overall
exterior dimensions of 31/4".times.5".times.23/4". Each well is
capable of holding <2 mL of liquid volume. By combining a 96 tip
liquid handler with block handling robots and sufficient heaters
with stirrers or shakers, this IFR method can be extended to
include arrays of many 96 well blocks simultaneously reaching
steady states for analysis. Thus, potentially, hundreds of reactors
could be simultaneously addressed.
[0077] The following sequence is representative of the actual IFR
method used in the present invention. The sequence is not intended
to be limited to the details described, since various modifications
and substitutions can be made without departing from the spirit of
the present invention.
[0078] For discussion purposes only, each reaction well contained
50 mg of resin beads and 200 .mu.L of phenol:acetone; the volume
increments of fresh feed/sample were 30 .mu.L.
[0079] Molten phenol was poured into the phenol reservoirs and
maintained at the appropriate temperature (.about.80.degree. C.).
The reactor well array (96-well deep-well micro-titre plate) was
loaded with the appropriate amount of resin beads (about 25-65 mg),
along with phenol (total liquid volume typically ranges between
about 15 0-400 .mu.L). Optionally, the resin beads underwent a
pre-treatment step to attach a promoter to the resin beads (in
other experiments, the promoter was not attached to the beads but
was, instead, included in the stock solutions). The reactor array
was placed into the Reacti-Therm III heating/stirring module and
maintained at the appropriate reaction temperature
(.about.60-90.degree. C.).
[0080] Stock solutions containing phenol, acetone, and possibly a
promoter were loaded into a 48-well deep-well micro-titre plate.
The ratio of acetone:phenol is an experimental variable, but a
typical experiment might involve the use of about 2-9% acetone (by
weight) in phenol. A 48-well plate (rather than a 96-well plate)
was used because each reaction system was run in duplicate.
Therefore, each of the 48 stock solution wells were used to feed
two separate reactor wells. The stock plates were clamped into the
heated block and maintained at an appropriate temperature
(typically .about.60.degree. C.).
[0081] The system reservoir was topped-off with an appropriate
solvent, which was either maintained at room temperature or heated
using a heating mantle. 96 empty well microtitre plates were placed
at the appropriate position on the liquid-handling robot's deck.
These plates were used for sample collection.
[0082] The probes were dipped into the first phenol reservoir to
rinse the system fluid (and other contaminants) off the outside of
the probes. The probes were then moved to the second phenol
reservoir and a small volume of phenol was simultaneously loaded
into each probe. The probes were pulled out of the phenol reservoir
and an air gap was put into each probe. The probes were then moved
to the stock block, and a (30 .mu.L) aliquot of fresh feed was
pulled into each probe. It should be noted that at this point, each
probe was in a different well of the stock block, so different
stock solutions can be loaded into the different probes.
[0083] The probes were then moved to the first row of the reactor
array, and the (30 .mu.L) aliquots of fresh feed were delivered to
the reactor wells. The probes were then moved to the rinse station,
and a volume (250 .mu.L) of system fluid was expressed through the
probes to rinse out the phenol and feed solutions.
[0084] All operational steps previously described were then
repeated for the second row of the 8 reaction wells, and then again
in the same fashion for the third through twelfth rows. At this
point, an increment of fresh feed has been delivered to all 96
reaction wells. Now the robot returns for another pass (described
as follows), this time removing 30 .mu.L aliquots of sample from
each reaction well. The probes may need to idle a while until the
appropriate time interval has passed before starting in on another
cycle.
[0085] The first step of the new cycle involved rinsing each probe
in the first phenol reservoir to remove any system fluid and
contaminants from the outside of the probe. Next, the probes were
then moved to the second phenol reservoir and a small volume of
phenol was simultaneously loaded into each probe. The probes were
pulled out of the phenol reservoir and an air gap was put into each
probe. The probes were then moved to the first row of 8 reaction
wells, and set at a pre-determined height above well bottom. The
robot was then programmed to attempt to remove more than 30 .mu.l
(about 45 .mu.L). This step serves as a level control and corrects
for systematic differences between aspiration and dispensing
volumes. In other words, if the robot is programmed to add 30 .mu.L
and, in this step, remove 30 .mu.L, systematic errors may occur
which lead to either a build up or loss of standing volume in the
reactor wells. This solution represents one way to overcome this
problem with liquid handling robots. The removed samples were
either deposited in a waste container or in one of the sample
blocks for analysis later. The probes were then moved to the rinse
station, and a volume (250 .mu.L) of system fluid was expressed
through the probes to rinse out the phenol and the sample aliquots.
The steps beginning with the initial rinsing step (to remove the
system fluid and contaminants) of each probe through the final
rinse step (to remove the phenol and sample aliquots) were then
repeated for the second row of the 8 reaction wells. The steps were
again repeated in the same fashion for the third through twelfth
rows.
[0086] At this point, a complete add and remove cycle has been
performed. This is typically accomplished within about 15 minutes.
This add and remove cycle is then repeated about 40 times
(.about.10 hours). This cycle is variable. The last add and remove
cycle was slightly different. After the samples had been removed
from the reaction wells, the probes were moved to the sample
collection micro-titre plates and the samples were delivered to the
appropriate row of wells for subsequent analysis. Then, the probes
were rinsed, and the procedure continued. As previously noted,
sampling for analysis can be done during any cycle and the time
course of the reactor can be measured.
[0087] Following delivery of the sample aliquots to the sample
collection micro-titre plates, the sample plates were removed from
the liquid-handler's deck and prepared for gas chromatographic
analysis.
[0088] All references described herein are incorporated by
reference in their entirety.
[0089] While the invention has been illustrated and described as
embodied in a method for high throughput chemical screening, it is
not intended to be limited to the details shown, since various
modifications and substitutions can be made without departing in
any way from the spirit of the present invention. For example,
various detection techniques may be incorporated into the method to
provide data at accelerated rates. Also, quite often an
intermediate time-point set of samples is collected in addition to
the end-point set of samples. In this instant, procedurally, the
add and removal cycle previously described can be inserted into the
operational procedure at any point in the experiment for which data
is required.
[0090] Further modifications and equivalents of the embodiments
herein disclosed may occur to persons skilled in the art using no
more than routine experimentation, and all such modifications and
equivalents are believed to be within the spirit and scope of the
invention as defined by the following claims.
* * * * *