U.S. patent application number 17/143814 was filed with the patent office on 2021-04-29 for solvent delivery system for liquid chromatography that maintains fluid integrity and pre-forms gradients.
This patent application is currently assigned to Waters Technologies Corporation. The applicant listed for this patent is Waters Technologies Corporation. Invention is credited to Steven J. Ciavarini, Keith Fadgen, Jeffrey W. Finch, Hongji Liu, Stanley P. Pensak, JR..
Application Number | 20210123891 17/143814 |
Document ID | / |
Family ID | 1000005329426 |
Filed Date | 2021-04-29 |
United States Patent
Application |
20210123891 |
Kind Code |
A1 |
Ciavarini; Steven J. ; et
al. |
April 29, 2021 |
SOLVENT DELIVERY SYSTEM FOR LIQUID CHROMATOGRAPHY THAT MAINTAINS
FLUID INTEGRITY AND PRE-FORMS GRADIENTS
Abstract
A solvent delivery subsystem for a chromatography device
performs relatively low pressure, high flow mixing of solvents to
form a gradient and subsequent high pressure, low flow delivery of
the gradient to the separation column. The mixing of the gradient
is independent and does not interfere with the gradient delivery.
To form the gradient, the outputs of an aqueous pump and an organic
pump are mixed to fill a storage capillary while a downstream point
from the storage capillary is vented to atmosphere. After gradient
formation, the vent to atmosphere is closed, the solvent delivery
system rises to high pressure, and only the aqueous pump runs for
gradient delivery. To maintain integrity of the fluid stream, the
solvent delivery system uses feed forward compensation and controls
at least one parameter selected from the group consisting of
pressure and flow in the conduit means to follow a gradual
ramp.
Inventors: |
Ciavarini; Steven J.;
(Natick, MA) ; Pensak, JR.; Stanley P.; (East
Walpole, MA) ; Finch; Jeffrey W.; (Gig Harbor,
WA) ; Fadgen; Keith; (Whitinsville, MA) ; Liu;
Hongji; (Grafton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Waters Technologies Corporation |
Milford |
MA |
US |
|
|
Assignee: |
Waters Technologies
Corporation
Milford
MA
|
Family ID: |
1000005329426 |
Appl. No.: |
17/143814 |
Filed: |
January 7, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14822527 |
Aug 10, 2015 |
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17143814 |
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12280585 |
Nov 14, 2008 |
9103814 |
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PCT/US2007/006679 |
Mar 16, 2007 |
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14822527 |
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60783610 |
Mar 17, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2030/326 20130101;
G01N 30/32 20130101; G05D 11/132 20130101; G01N 30/34 20130101 |
International
Class: |
G01N 30/34 20060101
G01N030/34; G01N 30/32 20060101 G01N030/32; G05D 11/13 20060101
G05D011/13 |
Claims
1-68. (canceled)
69. A solvent delivery system for a liquid chromatography system,
comprising: a first leg including a first pump for introducing a
first solvent into the first leg; a second leg including a second
pump for introducing a second solvent into the second leg; a mixing
tee positioned at a junction between the first leg and the second
leg for mixing the first solvent form the first leg with the second
solvent from the second leg; a controller comprising a processor
for providing open loop feed forward control of the first pump and
the second pump based on a parameter of stored energy in the liquid
chromatography system.
70. The solvent delivery system of claim 69, further comprising a
first pressure sensor for measuring pressure in the first leg and a
second pressure sensor for measuring pressure in the second leg,
wherein the first pressure sensor and the second pressure sensor
are configured to provide pressure measurements to the
controller.
71. The solvent delivery system of claim 69, wherein the parameter
is a ratio of compressibility for the first solvent and the second
solvent.
72. The solvent delivery system of claim 71, wherein the controller
is configured for controlling the first pump and the second pump
based on the ratio of compressibility.
73. The solvent delivery system of claim 69, further comprising a
first flow sensor for measuring flow rate in the first leg and a
second flow sensor for measuring flow rate in the second leg,
wherein the first flow sensor and the second flow sensor are
configured to provide flow rate measurements to the controller.
74. The solvent delivery system of claim 73, wherein the controller
is configured for determining a fluid capacitance of the first leg
and a fluid capacitance of the second leg, wherein the fluid
capacitance of the first leg is a product of a fluid
compressibility constant for the first solvent and a captive
fluidic volume in the first leg from the first pump to the first
flow sensor and wherein the fluid capacitance of the second leg is
a product of a fluid compressibility constant for the second
solvent and a captive fluidic volume in the second leg from the
second pump to the second flow sensor.
75. The solvent delivery system of claim 74, wherein the controller
is configured for generating a feedforward correction for the first
pump as a product of a disturbance value for the first leg and the
fluid capacitance of the first leg and the controller is configured
for generating a feedforward correction for the second pump as a
product of a disturbance value for the second leg and the fluid
capacitance of the second leg.
76. The solvent delivery system of claim 75, the disturbance value
for the first leg is an estimate and the disturbance value for the
second leg is an estimate.
77. The liquid chromatography system of claim 75, wherein the
disturbance value for the first leg is a measured value and the
disturbance value for the second leg is a measured value.
78. The solvent delivery system of claim 75, wherein the controller
is configured to generate a control signal for the first pump that
encodes a sum of the feedforward correction for the first pump and
a feedback correction for the first pump, where the feedback
correction for the first pump is a difference between a desired
flow and a measured flow as measured by the first flow sensor.
79. The solvent delivery system of claim 75, wherein the first pump
has a plunger and the second pump has a plunger, wherein the
controller is configured to correct the fluid capacitance for the
first leg based on position of the plunger of the first pump before
generating the feedforward correction for the first pump, and
wherein the controller is configured to correct the fluid
capacitance for the second leg based on position of the plunger of
the second pump before generating the feedforward correction for
the second pump.
80. The solvent delivery system of claim 69, further comprising
conduits or series restrictors in the first leg and/or the second
leg to provide passive fluidic decoupling between the first pump
and the second pump.
81. A method, comprising: pumping a first solvent with a first pump
in a first leg of a solvent delivery system; pumping a second
solvent with a second pump in a second leg of the solvent delivery
system; mixing the first solvent and the second solvent at a mixing
tee positioned at a junction of the first leg and the second leg;
controlling the first pump and the second pump with a controller
that provides open loop feed forward control of the first pump and
the second pump based on a parameter of stored energy in the liquid
chromatography system.
82. The method of claim 81, wherein the parameter is a ratio of
compressibility for the first solvent and the second solvent.
83. The method of claim 81, further comprising: using a first flow
sensor to measure flow rate in the first leg; using a second flow
sensor to measure flow rate in the second leg; and providing flow
rate measurements from the first flow sensor and the second flow
sensor to the controller.
84. The method of claim 83, wherein the controlling the first pump
and the second pump with the controller comprises the controller
determining a fluid capacitance of the first leg and a fluid
capacitance of the second leg, wherein the fluid capacitance of the
first leg is a product of a fluid compressibility constant for the
first solvent and a captive fluidic volume in the first leg from
the first pump to the first flow sensor and also comprises the
controller determining the fluid capacitance of the second leg is a
product of a fluid compressibility constant for the second solvent
and a captive fluidic volume in the second leg from the second pump
to the second flow sensor.
85. The method of claim 84, wherein the controlling the first pump
and the second pump with the controller comprises generating a
feedforward correction for the first pump as a product of a
disturbance value for the first leg and the fluid capacitance of
the first leg and is configured for generating a feedforward
correction for the second pump as a product of a disturbance value
for the second leg and the fluid capacitance of the second leg.
86. The method of claim 85, wherein the controlling the first pump
and the second pump with the controller comprises generating a
control signal with the controller for the first pump that encodes
a sum of the feedforward correction for the first pump and a
feedback correction for the first pump, where the feedback
correction for the first pump is a difference between a desired
flow and a measured flow as measured by the first flow sensor.
87. The method of claim 85, wherein the first pump has a plunger
and the second pump has a plunger and wherein the controlling the
first pump and the second pump with the controller comprises
correcting the fluid capacitance for the first leg with the
controller based on position of the plunger of the first pump
before generating the feedforward correction for the first pump,
and correcting the fluid capacitance for the second leg with the
controller based on position of the plunger of the second pump
before generating the feedforward correction for the second
pump.
88. The method of claim 81, further comprising placing conduits or
series restrictors in the first leg and/or the second leg to
provide passive fluidic decoupling between the first pump and the
second pump.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Patent
Application No. 60/783,610, filed Mar. 17, 2006 which is
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Liquid chromatography (LC) is an analytical technique in
which an analyte or sample is examined. A LC system typically has
one or more columns that are packed with a stationary phase
material. Generally, the term "column" refers to columns,
cartridges, capillaries and the like for performing separations of
a chromatographic nature. Columns are typically packed or loaded
with a stationary phase. This stationary phase can be particulate
or beadlike or a porousmonolith or a substantially inert material.
For the purpose of the present disclosure, the term "column" also
refers to capillaries which are not packed or loaded with a
stationary phase but rely on the surface area of the inner
capillary wall to effect separations.
[0003] A mobile phase material or solution mixed with the analyte
is pumped into the column and the stationary phase material
separates and isolates the analyte. The mobile phase material may
comprise any fluid, such as, liquids, gases, supercritical fluids
or mixtures thereof. Often the analyte elutes directly from the
column to an inline detector to generate a chromatogram.
[0004] Typically, the analyte is only available in minute
quantities so that extreme care must be taken not to waste even the
smallest amount. Consequently, LC systems are designed to operate
on minute samples or nano-flows with columns that are nano-sized
capillaries such as described in U.S. Pat. No. 6,299,767, which is
incorporated herein by reference.
[0005] Several systems have been developed to accurately and
efficiently deliver the mobile phase material or gradient. The
delivery of the gradient is often under demanding constraints such
as high pressure, e.g., high pressure liquid chromatography (HPLC)
systems. For example, U.S. Pat. Nos. 6,858,435 and 6,610,201
describe various techniques in which the solvent composition is
formed and delivered. First in, first out (FIFO) is a commonly used
technique for forming and delivering the gradient.
[0006] Despite the advances noted above, many inherent limitations
remain in nano-flow LC systems. Very commonly the gradient is
formed for real-time, immediate consumption by the column. As a run
may take an hour, this increases the difficulty of maintaining the
proper mixture of the gradient under challenging circumstances. In
particular, nano-flow LC systems utilize nano-scale flow
transducers.
[0007] However well-suited to the particular application,
nano-scale transducers have a limited dynamic range and are
detrimentally subject to thermal effects. Formation of the gradient
over a typical period, such as an hour, during which the
temperature can fluctuate results in a poor mixture consistency
because of the thermal drift of the transducer. Further, on a
nano-scale where the transducers may only hold 2 .mu.L of fluid,
the high pressure compression of the fluids generates erroneous
transducer readings which spoil the mixture. High pressure
operation is also at the edge of the tranducer's dynamic range
where noise levels can become unacceptable. In short, limited
range, noise and thermal drift associated with the transducers are
obstacles to meeting the desired performance.
[0008] For example, a commercially available nano-flow thermal
anemometry type transducer, having a calibration range of 0 to 5
uL/min, operating at an elution flow rate of 250 nL/min, requires
repeatable calibration performance of the organic pump down to 7.5
nL/min, for an initial chromatographic starting composition of 3%.
This is not an easy performance specification to achieve because
the noise level of the device can be as high as +/-5 nL/min, and
the thermal sensitivity to noise without thermal compensation can
be in excess of +/-4 nL/min per degree Celsius. Thus, a one degree
change of the instrument temperature can result in almost a 50%
composition error. In an effort to remedy this error, the typical
commercially available direct nano-flow LC system employs an
external thermal compensation scheme with a temperature sensing
element to compensate the flow transducer. Such attempts at
compensating for the affects of the thermal sensitivity greatly
increase the complexity of the LC system without addressing the
underlying problem.
[0009] Often, the bulk flow to the LC system is changed during
starting and stopping flow, changing flow rates and transitioning
flows between operations. During bulk or solvent flow change, it is
difficult to maintain the desired composition mixture across the
mixing point or node. For example, when the steady-state back
pressure of the LC system is very high (e.g., greater than 5,000
psi), the two fluid streams (e.g., solvent and analyte) have
significantly different fluid properties (i.e., aqueous and organic
solvents) before the mixing node. Due to inherent limitations, such
as flow transducers with relatively slow response times (i.e., time
constants of several seconds), the necessary feedback is not
provided quickly enough to allow the system to be responsive and
maintain the desired composition mixture.
[0010] Rapid pressure changes from say 10,000 PSI to 200 PSI can
cause critical and expensive components like the column to be
destroyed. At the very least, the cycles of compression and
decompression create apparent flow when none is actually occuring.
Also, the compressibility changes of fluid create false readings
because the transducers are inherently sensitive to such rapid
fluid density changes as the back pressure swings across the
typically large range of pressure, and to some extent, the
adiabatic heating effect of the fluids. Thus, LC systems that
regulate flow between multiple solvent pumps using feed back
control cannot maintain accurate measured flow due to the effects
of the rapid compressibility changes and heating on the nano-flow
transducers.
[0011] Another disadvantage of prior art LC systems is cross-flow
and back-flow contamination of the flow transducers across the
mixing node, which results in contamination of the LC system fluid
stream and a temporary loss of flow calibration to the transducers.
During rapid decompression or during the normal chromatographic
delivery of the gradient, as the viscosity of the fluid drops
(i.e., higher organic), prior art LC systems require flow to be
reversed back through the mixing node. To buffer the back-flow
contamination of the transducers, the connecting conduits between
the flow transducers and the mixing node are sized to accommodate
the decompression volume of the mixing tee. However, the additional
volume becomes lumped with that of the transducers, further
exacerbating the rapid compressibility change problems noted above.
Thus, conventional high-pressure mixing systems are very
susceptible to cross-contamination across the mixing node at very
high pressures.
[0012] Further, the cross-contamination across the mixing node can
create feedback instability between two flow transducers resulting
from the fluidic inter-active coupling. The cross-contamination
also increases the loss of maximum operating pressure, which is
attributed to the large parasitic pressure drops associated with
much larger decoupling restrictors required by high-pressure mixing
systems to passively stabilize the control interaction. Previous LC
systems have recognized these problems and proposed redundant
pumps, complex plumbing schemes and valves to isolate the gradient
formation pumps from the high-pressure portion of the LC system.
Such costly and complex solutions are not only undesirable but the
problems remain.
[0013] Still further, some LC applications require changing the
operational flow rate for a particular choice of column during an
injection run, e.g., sample trapping and 2-D chromatography. The
flow rate must be started and stopped between selection of each
column. Prior art systems have employed some means of valve
switching at essentially a no-flow condition to accomplish such
flow rate changes. The valve switching components deter from the
reliability of the LC system while increasing the expense and
complexity in an unfavorable manner.
[0014] Other systems have also been developed in an effort to
increase the sensitivity and/or collect more data from samples. For
example, U.S. Pat. No. 6,858,435 discloses LC analysis systems that
make use of a variable flow or peak parking to overcome the
difficulty a detector may have in adequately sensing the various
species with the sample liquid. When the LC analysis system detects
a peak of interest, the LC analysis system controls a
micro-switching valve to rapidly reduce the elution flow rate
(i.e., reduction in flow by 20 to 50 times). As a result, the
elution time of the column-separated compounds is extended to
enhance detection. After analysis, the LC analysis system restores
the normal elution flow rate. Again, the employment of additional
components to accomplish peak parking unfavorably increases the
expense and complexity of the system.
[0015] Another method to increase the efficiency of LC analysis is
to utilize microscale or nanoscale flow rates such as 0.025 to 100
ul/min flow rates. By using such flow rates, the LC analysis system
can produce ultra high sensitivity analysis. However, gradient
delay and dispersion become problematic. Further, sample loading
time and thereby the whole runtime become undesirably long.
[0016] As can be seen from the discussion above, closed-loop
feedback mechanisms have been developed for LC analysis systems.
However, there is a need for still better control and prior art
systems do not use feed-forward open-loop mechanisms. Feed-forward
is an approach to reacting to changes in a system to minimize or
prevent error.
SUMMARY OF THE INVENTION
[0017] In view of the above, sytems for delivering solvents to
chromatography devices that ovecome the aforementioned problems of
the prior art are needed.
[0018] The invention addresses the problems above and others by
providing systems and methods for delivering solvents to liquid
chromatography devices. The inventors have discovered that by using
a feed forward control strategy to compensate the effects of fluid
compressibility, with conventional closed-loop feedback control,
the ultimate preparation and delivery of the solvent can be greatly
improved.
[0019] In one embodiment, the subject technology is directed to a
system for delivering a gradient to a liquid chromatography device
having an injector that introduces the sample into a separations
column. The system includes a first leg having an aqeous pump
producing an aqeous output directed through a first inline pressure
transducer and a first flow transducer, a second leg having an
organic pump producing an organic output directed through a second
inline pressure transducer and a second flow transducer and a
processing device for controlling the legs. The processing device
includes a closed-loop feedback mode to generate a corrective
control signal based on a signal derived from at least one of the
transducers to overcome parasitic losses upstream from the at least
one of the transducers and an open-loop feed forward mode to
generate anticipatory control signals based on a parameter of
stored energy of the system, wherein the anticipatory control
signal calculates a compression flow based on a ratio of
compressibility between the aqeous output and the organic output,
and wherein the processing device can selectively operate each leg
in different modes.
[0020] In another embodiment, the subject technology is directed to
a fluidic chromatography instrument including a first leg having a
pump and transducers for monitoring pressure and flow output from
the pump, a second leg having a pump and transducers for monitoring
pressure and flow output from the pump, a controller for providing
instructions to each pump based on signals from the transducers,
and a node for mixing outputs from the first and second legs,
wherein the controller can provide a feed forward signal to the
first and second pumps based on an ability of the first and second
legs to store energy for controlling the compressibility of fluids
in the first and second legs to maintain composition control across
the node.
[0021] In one embodiment, the cycles of compression and
recompression are closely controlled by using a feed forward
algorithm and/or a linear valve to minimize fluidic disturbances.
Further, several components, such as pumps, when used smartly can
alleviate needs for additional components.
[0022] In another embodiment, an improved system for pre-forming a
gradient in a nano-flow solvent delivery system allows efficiently
creating and delivering the gradient. Still further, by adding
additional components, such as pumps, formerly serial actions can
be performed in parallel to reduce elution run and set up time.
[0023] Thus, in one aspect, the invention provides a method of
forming a gradient for a liquid chromatography system having a pump
that fills a storage capillary. The method includes the steps of
venting the storage capillary to atmosphere and running the pump at
relatively low pressure and higher flow rate to fill the storage
capillary until the gradient is formed therein. Preferably, an
optimized volume geometry of the storage capillary is sized by a
length and an inner diameter to minimize formation of backpressure
and gradient dispersion. In another embodiment, the volume capacity
of the storage capillary is sized to accommodate the gradient and
an overhead of transport volume necessary to move the gradient to a
separations column.
[0024] The pump may actually be an aqueous pump and an organic
pump, each pump having an output connected to a mixing node
intermediate the pumps and the storage capillary. During gradient
delivery, the organic pump is offline, the vent to atmosphere is
closed and the aqueous pump runs to deliver the gradient to a
separation column. To purge the storage capillary, the storage
capillary is vented to atmosphere and at least one of the pumps is
run to ready for forming another gradient.
[0025] Formation of the gradient with the storage capillary
essentially vented to atmosphere accomplishes three important
functions: 1) the formation back pressure is accurately controlled
by the geometry of the storage capillary, independent of the column
or other connected consumables; 2) the fluid in the storage
capillary is purged to waste to prevent upsetting the equilibrium
state of the column between injection runs; and 3) any leading or
trailing compositional aberrations bracketing the formed gradient,
due to starting and stopping the flow during formation, are
directed away from the primary fluid stream of the system, i.e.,
away from the analytical portion and the column therein.
[0026] Further, separation of gradient formation at low pressure
and isocratic delivery of the gradient at high operational
pressures fundamentally eliminates the interdependent coupling of
solvent mixture compositional accuracy to changes in flow rate,
compared to conventional approaches of high-pressure mixing and
delivery. In other words, the subject technology makes gradient
formation orthogaonal to delivery, which does not change the
gradient mixture during the run.
[0027] It is a further advantage of the subject technology to
provide a solvent delivery system that forms the gradient at high
flow rates closer to the transducer's full scale calibration,
thereby eliminating the need to extend the dynamic range of the
flow transducers far below the elution flow rate, which requires
extending the performance at or near the zero-flow calibration of
the transducers. By thus avoiding the transduer region that is very
susceptible to both noise and thermal drift, the need and
additional cost for thermal compensation and characterization of
the flow transducer are eliminated while gradient composition
accuracy is improved.
[0028] Another advantage of the subject technology is the short
time intervals to create the gradient, which practically eliminates
chromatographic retention time fluctuation due to thermal effects.
By forming the gradient in a short time interval, susceptibability
to temperature effects is removed. Accordingly, the need for
thermal compensation is also reduced or eliminated.
[0029] Another advantage of gradient formation at low pressure and
subsequent delivery by a single pump is the ease of maintaining the
desired composition mixture to across the mixing tee.
[0030] In another aspect, the inventoin provides a system for
providing a gradient to a nano-flow capillary liquid chromatography
device. The system includes an aqeous pump producing a first
output, a organic pump producing a second output mixed with the
first output to produce a solution and a processing device for
controlling the pumps. A storage capillary receives a formed
gradient from a portion of the solution. A fitting is connected to
the output of the storage capillary, wherein the fitting forms a
first outlet connected to the nano-flow capillary liquid
chromatography device and a second outlet. A valve connects to the
second outlet and is controlled by the processing device such that,
during formation of the gradient in the storage capillary, the
valve is open to direct resident fluid to waste while the aqeous
and organic pumps run.
[0031] Preferably, the system also has a first inline pressure
transducer and a first flow transducer for receiving the first
output and a second inline pressure transducer and a second flow
transducer for receiving the second output, wherein each transducer
is in communication with the processing device to provide
closed-loop feedback control. In one embodiment, the storage
capillary, the fitting and the valve are co-located in the
thermally managed compartment of the separations column.
[0032] Another advantage of the subject technology is to perform
gradient delivery by a single pump with the control means of having
both a pressure and a flow transducer that allows for very rapid
flow changes, return to steady-state flow operation, and
elimination of the serious mixture contamination and stability
control problems described above. As a result, the subject
technology easily accommodates the requirements of performing
variable flow or peak parking operation at very high pressures, as
compared with conventional high-pressure mixing systems. Further,
the need for redundant pumps, complex plumbing schemes and valves
in an effort to have the desired isolation of the gradient
formation pumps from the high-pressure portion of the system is
eliminated.
[0033] In another aspect, the invention provides a solvent delivery
subsystem for a LC device. The solvent delivery subsystem includes
a first pump producing a first output, a first transducer connected
to receive the first output for monitoring a parameter thereof, a
second pump producing a second output mixed with the first output
to produce a solvent mixture, a second transducer connected to
receive the second output for monitoring a parameter thereof, a
mixing node for combining the first and second outputs and
restrictive conduits between each pump and the mixing node to
provide passive fluidic decoupling between the first and second
pumps to stabilize control interactions across the mixing node.
Preferably, the conduits are capillary restrictors and upstream
from the first and second transducers.
[0034] A further advantage of the subject technology is to prevent
cross-flow and back-flow contamination of the flow transducers
across the mixing node. Yet a further advantage is to avoid the
feedback instability between flow controller. Still a further
advantage is to reduce the loss of maximum operating pressure
attributed to the large parasitic drops of the larger decoupling
restrictors by alleviating the need the for large decoupling
restrictors.
[0035] Another aspect of the invention provides a system for
delivering a gradient to a liquid chromatography device having an
injector that introduces the sample into a separations colums. The
system includes an aqeous pump producing a first output directed
through a first inline pressure transducer and a first flow
transducer, a organic pump producing a second output directed
through a second inline pressure transducer and a second flow
transducer and a processing device for controlling the pumps in a
closed-loop feedback mode based on a signal from at least one of
the transducers to overcome losses upstream from the
transducer.
[0036] Preferably, the system has a first fitting for mixing the
first and second output to produce a third output and a storage
capillary for forming a gradient. The gradient is a portion of the
third output. The storage capillary is sized to minimize
backpressure and dispersion. A second fitting is connected to the
storage capillary, wherein the second fitting forms two outlets,
the first outlet being connected to the nano-flow, capillary liquid
chromatography device. A valve is connected to the second outlet of
the second fitting and controlled by the processing device. Upon
forming the gradient in the storage capillary, the valve is open to
direct resident fluid to waste while the aqeous and organic pumps
run. During delivery of the gradient to the nano-flow capillary
liquid chromatography device, only the aqeous pump runs and the
organic pump is offline.
[0037] Still another embodiment of the subject technology is
directed to a system for changing an operational flow rate of
delivery of a gradient to suit a first column of a nano-flow
capillary liquid chromatography device. The system includes a pump
producing an output directed through a pressure transducer and a
flow transducer, a storage capillary for forming the gradient from
a portion of the output and a processing device for controlling the
pump and receiving signals from the transducers.
[0038] The processing device has a memory storing an instruction
set and a processor for running the instruction set. The processor
is operative to store a system pressure measurement from the
pressure transducer at an end of an injection when a second column
of the nano-flow capillary chromatography device is
re-equilibriated. The processor is also opeative to stop flow to
the nano-flow capillary chromatography device, form a new gradient
particularly suited to the first column, set a target pressure
equal to the system pressure measurement, receive and store a flow
rate selected by a user for the first column, and use the pump
under closed-loop pressure control with feedback from the pressure
transducer to bring a system pressure to the first target pressure.
Upon reaching the first target pressure, the processing device
transitions the pump to closed-loop flow control with the flow
transducer as feedback and the flow rate as a target flow rate to
recommence delivery of the new gradient by running the pump.
[0039] Preferably, the system also has a second pump producing an
output directed through a second pressure transducer and a second
flow transducer, a mixing node connected to the storage capillary
for combining the outputs of the pumps, and a valve connected to
the storage capillary. The valve is controlled by the processor for
venting the storage capillary to a pressure below that of an
operating pressure of the system.
[0040] It is another advantage of the subject technology to
accomplish changing the flow rate in a simple, efficient and quick
manner without adding to the complexity of the system. Yet another
advantage is the ability to overcome the long time constant in
trapping applications.
[0041] Still another aspect of the invention provides a solvent
delivery system for a chromatography device having a short trapping
column in series with a restrictive column. The solvent delivery
system includes a first pump producing a first output, a pressure
transducer and a flow transducer connected to receive the first
output. A second pump produces a second output mixed with the first
output to produce a solvent mixture. A second pressure transducer
and a second flow transducer are connected to receive the second
output for monitoring a parameter thereof. A mixing node combines
the first and second outputs. A controller operates the pumps with
closed-loop feedback. The controller is programmed to use the
pressure transducers for the closed-loop feedback in a
pressure-control mode, use the flow transducers for the closed-loop
feedback in a flow-control mode, use both pumps in the flow-control
mode to form a gradient, use only the first pump to deliver the
gradient, stop flow by rapidly decompressing the short trapping
column in series with the restrictive column using the
pressure-control mode, set a reference pressure set point to zero
and commence operation of the first pump to overcome a long time
constant of the short trapping column in series with the
restrictive column.
[0042] In yet another apsect, the invention provides a method for
peak parking (sometimes referred to as variable-flow) in a liquid
chromatography system including the steps of pre-forming a gradient
from a mixture, controlling a flow rate by using a flow transducer
for closed-loop feedback, monitoring a delivery pressure by using a
pressure transducer. Based upon a signal to reduce the flow rate
based on an elution peak of interest, a target pressure is
calculated based on the delivery pressure. The flow rate is
controlled by using the pressure transducer as closed-loop feedback
with the target pressure as a set point.
[0043] Embodiments of the present invention are also directed to
methods and apparatus for controlling the composition or
maintaining the integrity of a fluid mixture. Differences in the
compressibility of compounds forming a mixture may cause the
compounds to flow differently. For example, organic compounds are
typically more compressible than aqueous solutions. If flow is
stopped suddenly in a conduit having a branch containing an organic
compound and a branch having an aqueous compound, the branch with
the organic compound may experience greater changes in volume
associated with pressure changes and may store energy differently.
These changes in volume and stored energy results in mixtures that
deviate from the desired composition.
[0044] One embodiment directed to a device, for use in the context
of a fluid stream being conveyed in conduit means under a range of
pressure comprising at least one operating pressure and one low
pressure and flow rates. The conduit means is in fluid
communication with pump means for propelling the fluid under
pressure. The conduit means is in fluid communication with valve
means, and the valve means has at least a first position and a
second position. In the first position the fluid flows in the
conduit means, and in the second position fluid does not flow in
the conduit means. The device comprises control means and ramp
means. The control means is in signal communication with the valve
means and in signal communication with ramp means. The ramp means
is for controlling at least one parameter selected from the group
consisting of pressure and flow in the conduit means. The ramp
means performs at least one of the following functions: (i) in
response to the placing or anticipated placing of the valve means
in the second position, while said conduit means is at said one or
more operating pressures, the control means commanding said ramp
means to decrease the selected parameter in said conduit means in a
first ramp until such low pressure is attained; and (ii) in
response to the valve means assuming the first position or in
anticipation of the valve means assuming the first position, to
increase the selected parameter until the one or more operating
pressures is attained in a second ramp.
[0045] As used herein, the term "pump means" refers to any means
for propelling a fluid. These means comprise serial and parallel
pumps, turbine pumps, syringe pumps, peristaltic pumps,
electrokinetic pumps, pneumatic amplifiers and fluids propelled
through compressed storage devices. The term "fluid communication"
refers to fluid connections of a plumbing type. The term "valve
means" is used to denote any device capable of interrupting the
flow of fluid. Chromatographic instruments have sample injectors
that are a special form of a valve.
[0046] As used herein, the term "control means" refers to
computers, computing processing units (CPUs), micro-controllers,
digital signal processors, (DSPs), servers, analog devices
programmed with suitable firmware or software. Analytical
instruments often have computers and CPUs controlling aspect of the
operation. Software to control single instruments and multiple
instruments through local or centralized computers are well known
in the art. The term "signal communication" refers to electrical
connections or circuits, optical connections, wireless
communications through radio waves, internet connections, and other
means by which equipment may communicate.
[0047] Another embodiment of ramp means comprises pump control
means. The pump control means can be a separate computer, computing
processing unit (CPU), micro-controller, DSPs, server or the same
control means controlling other functions. The pump control means
is for commanding one or more pumps to place at least one fluid in
the conduit means under a pressure in at least one of the first
ramp or second ramp. In order to reduce pressure or reduce flow,
the pump control means, particularly with small diameter conduit
means, may need to reverse in pumping direction.
[0048] One embodiment of the present invention features valve means
in the form of a valve for controlling flow from a trapping column.
Another embodiment of the present invention features valve means in
the form of a sample injection means. Sample injection means are
known in the art under several names, such as sample managers,
autosamplers, sample modules and the like. Sample injection means
are a form of a multi-port valve with a loop of conduit in which
the sample is placed. With the sample placed in the loop the
multi-port valve is placed with the loop in fluid communication
with the conduit means in fluid communication with pump means. The
sample injection means has a first position and a second position.
In the first position, the sample injection means receives a fluid
sample at a range of pressure comprising atmospheric pressure to
the low pressure. In the second position, the sample injection
means is in fluid communication with the conduit means for placing
the fluid sample in therein.
[0049] One embodiment of the present device has features drawn to
the sample injection means. The sample injection means is in fluid
control with a pressure source for placing a pressure in the sample
means. The pressure source is in signal communication with control
means, and control means commands the pressure source to place the
sample injection means under atmospheric or an initial pressure for
receiving the sample. And, after the sample is received, the
control means commands the pressure source to increase pressure to
the low pressure corresponding to the pressure in the conduit
means.
[0050] Preferably, the device further comprises at least one
pressure sensor in signal communication with the control means. The
pressure sensor is in fluid communication, that is, measures the
pressure of at least one of the pump means, conduit means and valve
means.
[0051] Preferably, the device comprises pump means. One embodiment
of the present invention features pump means comprising at least a
first pump for a first solvent and a second pump for a second
solvent. The conduit means comprises at least one first branch in
fluid communication with said first pump and a second branch in
fluid communication with said second pump. The first branch and
second branch are in fluid communication at a tee fitting wherein
said first solvent and second solvent form mixtures which comprise
the fluid.
[0052] Preferably, one pump is selected from said first and second
pump for control ramped to pressure. Preferably, at least one pump
is selected for control based on flow. With respect to control
ramped to flow, the flow of such pump is preferably controlled to
track the flow of the pump ramped to pressure, preferably
compensating further for the compressed volume of fluid. That is,
the pump tracking flow tracks the flow for the solvent for which is
expected to pump as a percentage of the total fluid plus an
additional volume of fluid representing the difference between the
compressed and uncompressed volume of the solvent. Preferably, the
pump ramped to pressure is for an aqueous solvent. And, the pump
ramped to flow is for an organic solvent.
[0053] And, preferably the device has at least one flow sensor in
signal communication with the control means and in fluid
communication with pump means, conduit means or valve means. The
flow sensors allow the matching of flow. As used herein, flow
sensors may also comprise stepped motors which relate to piston
movements which can be related to flow.
[0054] The device of the present invention features a ramp means
which coordinates the opening and closing of valve means with a
pressure ramp in the conduit means. The ramp is gradual in the
sense that the increase of decrease in pressure is not immediate,
having a slope of pressure over time of not greater than
approximately 10,000 pounds per square inch (psi) per second. More
preferably, the slope is in the range of 10 to 1,000 psi per
second, and, more preferably, 100 to 500 psi per second.
[0055] A further embodiment of the present invention is drawn to a
method of maintaining the integrity of a fluid. The method is for
use in the context of a fluid stream being conveyed in conduit
means under a range of pressure comprising at least one operating
pressure and one low pressure. The conduit means is in fluid
communication with pump means for propelling the fluid under
pressure. The conduit means is in fluid communication with valve
means, and the valve means has at least a first position and a
second position. In the first position the fluid flows in the
conduit means, and in the second position fluid does not flow in
the conduit means. The method comprises the step of controlling at
least on parameter selected from the group consisting of pressure
and flow in the conduit means. The parameter is controlled in at
least one of the following steps:
[0056] (i) in response to the placing or anticipated placing of the
valve means in the second position, while the conduit means is at
said one or more operating pressures, to decrease the selected
parameter in the conduit means in a first ramp until the low
pressure is attained; (ii) in response to the valve means assuming
the first position or in anticipation of the valve assuming the
first position while the conduit means is at the low pressure to
increase the selected parameter until one or more operating
pressures in a second ramp is attained.
[0057] Thus, embodiments of the present invention are directed to
controlling the composition or maintaining the integrity of a fluid
mixture. Differences in the compressibility of compounds forming a
mixture may cause the compounds to flow differently. These changes
in volume and stored energy results in mixtures that deviate from
the desired composition. Embodiments of the present invention
gradually reduce the stored energy of the conduits, pumps and
valves prior to disrupting the flow. These and other features of
the present invention will be apparent from viewing the drawings
and studying the text of the Detailed Description that follow.
[0058] Another embodiment of the subject technology is directed to
a LC instrument including an injection valve for creating a
gradient, an analytical column in fluid communication with the
injection valve, a pump connected to the injection valve for urging
the gradient towards the analytical column and a device
intermediate the injection valve and analytical column for storing
the gradient.
[0059] It should be appreciated that the present invention can be
implemented and utilized in numerous ways, including without
limitation as a process, an apparatus, a system, a device, a method
for applications now known and later developed or a computer
readable medium. These and other unique features of the system
disclosed herein will become more readily apparent from the
following description and the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0060] FIGS. 1A and 1B are a somewhat schematic block diagram
illustrating a HPLC instrument using a solvent delivery susbsytem
that utilizes feed-forward principles in accordance with the
subject technology, wherein matching instructions are present to
illustrate how to properly connect FIGS. 1A and 1B.
[0061] FIG. 2 is a flow diagram of a process for delivering a
mixture in the instrument of FIGS. 1A and 1B in accordance with the
subject technology.
[0062] FIG. 3 is a timing diagram representing flow and pressure
changes during a cycle of operation in the context of the HPLC
instrument of FIGS. 1A and 1B.
[0063] FIGS. 4A and 4B are a somewhat schematic block diagram
illustrating a HPLC instrument using a solvent delivery susbsytem
that utilizes feed-forward principles in accordance with the
subject technology, wherein matching instructions are present to
illustrate how to properly connect FIGS. 4A and 4B.
[0064] FIG. 5 is a somewhat schematic representation of the control
means of FIGS. 4A and 4B in greater detail to illustrate a flow
control strategy in accordance with the subject technology.
[0065] FIG. 6 is an electrical circuit analog to the instrument of
FIGS. 4A, 4B and 5 in accordance with the subject technology.
[0066] FIG. 7 is a somewhat more detailed version of the instrument
of FIGS. 4A and 4B is shown to more particularly illustrate the
usage of feed forward compensation in accordance with the subject
technology.
[0067] FIG. 8 is another more detailed version of an LC instrument
shown to to more particularly illustrate particular components of a
feed forward signal in accordance with the subject technology.
[0068] FIG. 9 is another electrical circuit analog to an LC
instrument of having a system or load flow Is to provide energy or
charge Cs to the load in accordance with the subject
technology.
[0069] FIG. 10 is still another more detailed version of an LC
instrument shown to more particularly illustrate a hybrid approach
using a feed forward signal in accordance with the subject
technology.
[0070] FIG. 11 is a somewhat schematic block diagram illustrating a
direct-flow nano-scale HPLC system using a solvent delivery
susbsytem that preforms gradients in accordance with the subject
technology.
[0071] FIG. 12 is a flow diagram of a process for forming and
delivering a gradient in the system of FIG. 1 in accordance with
the subject technology.
[0072] FIG. 13 is another flow diagram of a process for avoiding a
slow flow startup when performing a series of injection runs using
different columns in accordance with the subject technology.
[0073] FIG. 14 is a somewhat schematic block diagram illustrating a
direct-flow nano-scale trapping system using a solvent delivery
susbsytem in accordance with the subject technology.
[0074] FIG. 15 is a somewhat schematic block diagram illustrating
another direct-flow nano-scale trapping system using a solvent
delivery susbsytem with an additional pump in accordance with the
subject technology.
[0075] FIG. 16 is another flow diagram of a process for peak
parking in accordance with the subject technology.
[0076] FIG. 17 is a somewhat schematic block diagram illustrating a
LC instrument using a gradient storage device without a vent valve
in accordance with the subject technology.
[0077] FIG. 18 is a somewhat schematic block diagram illustrating
another LC instrument using a gradient storage device with a vent
valve in accordance with the subject technology.
[0078] FIG. 19 is a somewhat schematic block diagram illustrating
still another LC instrument using a gradient storage device with a
vent valve, binary pump and isocratic pump in accordance with the
subject technology.
DETAILED DESCRIPTION OF CERTAIN ILLUSTRATIVE EMBODIMENTS
[0079] The present invention overcomes many of the prior art
problems associated with delivering solutions to nano-flow
capillary LC instruments. The present disclosure maintains the
integrity of a fluid mixture by recognizing when undesirable mixing
may occur and employing pump settings to prevent such mixing. The
present disclosure also illustrates pre-forming gradients a low
pressure and high flow in order to increase throughput of LC
instruments. The advantages, and other features of the system
disclosed herein, will become more readily apparent to those having
ordinary skill in the art from the following detailed description
of certain illustrative embodiments taken in conjunction with the
drawings which set forth representative embodiments of the present
invention.
[0080] All relative descriptions herein such as upstream,
downstream, left, right, up, and down are with reference to the
Figures, and not meant in a limiting sense. Additionally, for
clarity, common items such as filters, conduits and
interconnections have not been specifically included in the Figures
as would be appreciated by those of ordinary skill in the pertinent
art. Unless otherwise specified, the illustrated embodiments can be
understood as providing exemplary features of varying detail of
certain embodiments, and therefore, features, components, modules,
elements, susbsytems and/or aspects of the illustrations can be
otherwise combined, interconnected, sequenced, separated,
interchanged, positioned, and/or rearranged without materially
departing from the disclosed systems or methods.
Linear Valve Embodiments
[0081] Referring to FIGS. 1A-2B, a direct-flow nano-scale HPLC
instrument 111 that uses a linear valve 127 in accordance with the
subject technology is shown. The linear valve 127 allows the HPLC
instrument 111 to efficiently transition between the desired
operating pressures. It is envisioned that the HPLC instrument may
operate at pressures exceeding 5,000 pounds per square inch (PSI)
as recently instruments have been introduced into the marketplace
capable of operation at 15,000 PSI.
[0082] Referring to FIGS. 1A and 1B, the instrument 111 has pumps
115a, 115b (collectively a binary pump 115) for moving respective
solvents A, B contained in respective reservoirs 135a, 135b through
conduits. The pumps 115a, 115b may comprise serial and parallel
pumps, turbine pumps, syringe pumps, peristaltic pumps,
electrokinetic pumps, pneumatic amplifiers and fluids propelled
through compressed storage devices. Typical pumps used in
chromatographic applications are serial pumps powered by stepper
motors. These pumps 115a, 115b are available from several venders.
Waters Corporation of Milford, Mass. sells suitable pumps under the
trademarks ALLIANCE.RTM., ACQUITY.RTM., and 600.TM. model
pumps.
[0083] The conduits interconnect the pumps 115a, 115b with the
reservoirs 135a, 135b as well as the other components of the HPLC
instrument 111. These solvents may comprise any solvents use in
chromatographic separations. Typical solvents may comprise a
aqueous solution as one solvent (e.g., water) and an organic
solvent (e.g., methanol, acetonitrile and the like). The conduits
include structures like tubes, pipes, capillaries and microfluidic
structures for conveying a fluid stream under a wide range of
pressures. Low pressure may be atmospheric pressure but may also be
a value several hundred or more above atmospheric pressure. For
simplicity, the conduits are typically unlabled and represented as
lines interconnecting the components in the Figures.
[0084] Typically, the conduits are stainless steel or silica,
however, other materials can be readily substituted. These
materials include by way of example, without limitation, brass,
aluminum, copper, titanium, ceramics, and plastics. The conduits
may comprise any size; however, embodiments of the present
invention have particular utility where the conduits are of a small
internal diameter. For example, without limitation, the conduits or
portions thereof are stainless steel tubing of 0.005 to 0.010 inch
diameter. Other portions of the conduits are silica capillary of
approximately 25 micrometer. In one embodiment, the conduits convey
flow rates in an approximate range of 50 nanoliters per minute to 5
microliters per minute when performing analytical processes and 4
microliters per minute to 20 microliters per minute when isolating
a material from a sample.
[0085] As illustrated, the pressure and flow of fluid from the
pumps 115a, 115b can be monitored by pressure sensors and flow
sensors, such as pressure transducer 131a and flow transducer 133a
for pump 115a, and pressure transducer 131b and flow transducer
133b for pump 115b. The pumps 115a, 115b and transducers 131a,
131b, 133a, 133b are in signal communication with control means
125. Signal communication is indicated by dotted lines. For piston
type pumps, the control means 125 commands the pumps 115a, 115b to
move the pistons forward or backward through signals. As a result,
the pumps 115a, 115b use closed-loop feedback to accurately control
pressure and/or flow.
[0086] The control means 125 refers to computers, computing
processing units, micro-controllers, digital signal processors,
servers, analog devices programmed with suitable firmware and the
like. Computers used in analytical instruments are well known to
and are available from several common vendors. The control means
125 is in signal communication with pressure sensors or transducers
131a and 131b and flow sensors or transducers 133a and 133b to
monitor and control the pressure and flow in accordance with FIG.
3. Commands are programmed into the firmware of control means 125
or into operating software through toolkits provided by software
manufacturers. Programming of this nature is routine by individuals
skilled in such art. Instrument control software is available from
several vendors such as Waters Corporation of Milford Mass. under
the trademarks EMPOWER.TM. or MILLINNIUM.RTM..
[0087] Still referring to FIGS. 1A and 1B, the outputs of the pumps
115a, 115b flow into a tee fitting 123 via conduit means 117a-117g.
In effect, solvent A and solvent B come together at the tee fitting
123 to form a mixture. The mixture then enters a linear valve 127,
which acts as ramp means. The term "ramp means" refers to means for
reducing or increasing the selected parameter gradually over time.
A ramp period is the period in which these changes are effected. A
ramp period is typically at least five seconds but may be much
smaller or much larger depending upon the particular application.
The ideal ramp period is based on the size of the fluid volumes,
flow rates and pressures. Although shown as an explicit item, it is
envisioned that the instrument 111 could be configured and operated
to include ramp means without having the discrete linear valve
127.
[0088] The linear valve 127 can selectively convey the mixture to
waste or a sample injector valve 119 for use in a trap column 165
and trap valve 121. The linear valve 127 has a range of openings
and is capable of decreasing and increasing the pressure in the
conduits by shunting the flow to waste or recycle. The linear valve
127 is in signal communication with control means 125 and receives
commands to open or close thereby. Linear valves 127 are known in
the art and are available from several vendors such as Valco of
Cincinnati, Ohio.
[0089] Linear valves 127 are characterized in that the flow can be
controlled in a gradual manner. Thus, the pressure and flow of the
device 111 can be controlled in the manner illustrated in FIG. 3.
Preferably, the linear valve 127 is controlled by a specific
portion 129 of the control means 125. The linear valve control
portion 129 can be a separate computer, computing processing unit
(CPU), server or the same control means 125 controlling other
functions, as illustrated.
[0090] When the linear valve 127 is set to allow flow forward in
the instrument 111, the mixture is conveyed through the linear
valve 127 to a sample injector valve 119 for use in a trap column
165 in combination with a trap valve 121. It is desirable to
control the composition of the mixture such that the components of
the sample are released in a reproducible manner. The tracking of
pressure and flow maintains the integrity of the fluid in the
conduits of the instrument 111.
[0091] Generally, "sample injector" refers to a form of valve and
conduits used to bring a section of conduit holding a sample into
fluid communication with conduits upstream of a column. The term
"valve" refers to means to control, restrict or stop flow. Sample
injectors normally comprise multiport valves and a loop (not shown)
of conduit for holding a sample. The sample injector valve 119 is
in fluid communication with a pressure source 151 which places
sample injector valve 119 under pressure. This pressure can be
matched by the control means 125 and linear valve 127. The pressure
source 151 may comprise any pump or source of compressed fluid.
Preferably, the pressure source 151 is a syringe pump (not shown)
known in the art and available from several vendors. The term
"pressure source" refers to any pump, syringe or compressed air or
fluid tank and the like. Sample injectors 119 typically have
syringes for withdrawing or aspirating samples.
[0092] The sample injector valve 119 and the trap valve 121 each
have at least a first position and a second position. In the first
position, the fluid flows and in the second position fluid does not
flow. Sample injector valves 119 are sold by several venders as
component parts or as part of an overall separation module. One
sample injector valve is sold as a component of a separation module
by Waters Corporation of Milford Mass. under the trademarks
ACQUITY.TM. and ALLIANCE.RTM.. Trapping valves are sold several
venders including Valco. The sample injection valve 119 places a
sample in a loop of conduit (not shown).
[0093] The linear valve 127 controls at least one parameter
selected from the group of flow and pressure, in the conduit means
117a-117g, in response to the placing or anticipated placing of the
sample injector valve 119 and trapping valve 121 in the second
position, while the instrument 111 is at one or more operating
pressures. The trapping valve 121 can also selectively direct flow
to waste or to an analytical column 2657 and a detector 169 without
perturbations in the solvent solutions. Thus, compounds of interest
captured on trap column 2655 can be effectively analyzed.
[0094] In one embodiment, one pump is selected from said first pump
115a and second pump 115b for control, ramped to pressure and the
other is selected for control, ramped to flow. With respect to
control ramped to flow, the flow of such pump is preferably
controlled to match the flow of the pump ramped to pressure.
Preferably, the pump ramped to pressure is for an aqueous solvent.
And, the pump ramped to flow is for an organic solvent. For
example, solvent A is an aqueous solvent. Pump 115a is commanded by
control means 125 to follow ramp 141a in FIG. 3. Pump 115b tracks
pump 115a with respect to flow on ramp 14 lb. The control means 125
commands pump 115b to maintains the correct mixture of solvent A
and solvent B by coordinating the delivery of pump 115b to
correspond to the delivery of pump 115a for the mixture
composition. In addition, pump 115b pumps or withdraws or adds the
compressed or uncompressed volume represented by the solvent B in
the conduit means 117a-117g and the pump 115b as pump 115b volume
changes.
[0095] Turning now to FIG. 2, a method for a cycle of operation of
the instrument 111 of FIGS. 1A and 1B is shown. Most generally, the
method is for use in the context of a fluid stream being conveyed
in conduits under a large range of pressure and/or flow. In HPLC,
the method is drawn to maintaining the integrity of a fluid despite
the disparate conditions that are necessary during the different
processing steps of trapping and elution. In FIG. 3, a timing
diagram illustrating a pressure profile 170 and a flow profile 172
during the method of FIG. 2 is shown with the regions corresponding
to the steps of FIG. 2 noted.
[0096] Referring to FIGS. 2 and 3, at step S1, the controller 125
records the current pressure and fluidic load conditions of the
instrument 111. Typically, the current conditions are those desired
during the previous elution run (e.g., high pressure and low flow).
Thus, the trap valve 121 is set so that flow is directed to the
analytical column 2657. In order to transition from elution run
conditions to trapping conditions (e.g., relatively low pressure
and high flow), great care must be taken to release the stored
energy stored in the instrument fluid without spoiling the
integrity of mixture by sloshing and backflow. Or even worse, if
the backflow becomes excessive, damage to the instrument components
like the column 2657 can occur.
[0097] FIG. 3 also indicates that current conditions may be those
associated with instrument start up as denoted by the cross-hatched
region 174. Region 174 shows that for start up conditions during an
initial portion S1a of step S1, the flow is zero but is ramped up
to analytical flow. In other words, the cross-hatching region 174
is an indication of the alternative lower flow profile 172 from
start up conditions until the instrument 111 can ramp to initial
gradient flow and composition.
[0098] Preferably during portion S1a, the instrument 111 also
advantageously loads the sample into the loop of the inject valve
119. The sample is loaded by setting the inject valve 19 to load
and using the pressure source 51. Further, the instrument 111
downloads the setup parameters for the next run. These setup
parameters are provided to the control means 125.
[0099] During the remaining portion S1b of step S1, the analytical
flow has been reached and, thus, the flow profile is consistent
between the two alternative approaches. Further, the instrument 111
stores the current parameters for the next run. These current
parameters of pressure and fluidic load, R, are provided to the
control means 125 as described below with respect to step S7.
[0100] At step S2, the instrument 111 starts the decompression ramp
to enable trapping conditions. The control means 125 commands the
linear valve 127 to decrease pressure in a first ramp 41a with
respect to pressure. Simultaneously, the control means 125 commands
the pump 115a to zero pressure and the pump 115b to zero flow
preferably with feed forward compensation (e.g., a hybrid
decompression ramp 141a). The term "feed forward compensation"
generally means open-loop compensation as will be described in more
detail below. Preferably, step S2 lasts about 30 seconds.
[0101] At step S3, the instrument 111 is at a low pressure and flow
condition 143a. Although this low pressure 143a can be atmospheric
pressure, as illustrated, the low pressure 143a can also be well
above atmospheric to correspond to a pressure in the sample
injector valve 119. To accomplish this ramp 141a, the linear valve
127 vents to waste and the control means 125 operates both pumps
115a, 115b set to zero flow. In effect, the energy stored in the
compressed fluid is released without or at least minimizing
backflow and sloshing. The pumps 115a, 115b become synchronized.
Preferably, step S3 lasts about 5 minutes.
[0102] At step S4, the instrument 111 readies for the trapping
process by directing the trap valve 121 to the trapping position,
i.e., the trap valve 121 directs flow to waste. Both pumps 115a,
115b ramp to the desired trapping conditions of relatively low
pressure and high flow as illustrated by ramps 145a, 145b,
respectively. To accomplish the ramps 145a, 145b, the control means
125 commands the linear valve 127 to increase pressure in the
conduits up to the operating pressure 147a and flow 147b.
[0103] Depending upon the flow generated by each pump 115a, 115b,
the mixture exiting the tee 123 can be controlled. The control
means 125 also readies the inject valve 119 to commence injection
of the sample in order to move the sample from the loop to the trap
column 165. Preferably, step S4 lasts about 30 seconds.
[0104] At step S5, the instrument 111 reaches equilibrium at the
trapping pressure 147a and trapping flow 147b with the desired
composition of the mixture. Preferably, instrument 111 delays for
about 1 minute to allow full equilibrium prior to commencing
injection of the sample. Upon reaching equilibrium, the sample is
moved from the loop to the trapping column 165 by setting the
inject valve 119 to the injection position. Because of the
relatively higher flow 147b, the time to transfer the sample can be
fast, on the order of minutes. Preferably, the user selects the
time period of trapping to insure that the sample is effectrively
moved to the trap column 165. In one embodiment, step S5 lasts
about 5 minutes.
[0105] At step S6, hybrid decompression occurs after the trapping
operation is complete. Although less than during an elution run,
the significant energy storyed in the instrument 111 needs to be
efficiently and effectively dissipated without mining the integrity
of the mixture in the conduits. The hybrid decompression includes
setting the pressure of pump 115a to zero while setting the flow of
pump 115b to zero preferably with feed forward. As a result, the
pressure declines to about atmospheric as shown by ramp 149a and
the flow moves to zero as shown by ramp 149b.
[0106] Again, to achieve these ramps 149a, 149b, the control means
125 commands the linear valve 127 to gradually vent to atmosphere.
The inject valve 119 is set to the load position, i.e., the loop
for holding the sample is removed from the fluid path to reduce the
amount of overhead that needs dissipation and subsequent
recompression. The trap valve 121 closes off to waste and directs
flow to the analytical column 2657. Upon reaching the minimal flow
and pressure conditions at the end of step S6, the instrument 111
can adjust settings to redirect fluid flow and the like while
maintaining the integrity of the fluid therein. Preferably, step S6
lasts about 30 seconds.
[0107] At step S7, the instrument 111 performs hybrid recompression
to accomplish an elurtion run without incurring an undesirably long
recompression time because of the relatively large volume of stored
energy required to compress the fluid stream back up to the
operational pressure. Prior to starting the hybrid recompression,
the control means 125 acquires the pressure and fluidic load
parameters stored at step S1b.
[0108] If the pressure and flow are the same as was previously
used, at least initially, the instrument 111 attempts to return to
the previous values. If the pressure and flow are different, the
instrument 111 extrapolates new settings based upon the stored
value of fluidic resistance R. For example, if the step S1b
parameters were a system pressure Ps of 9,000 PSI at a set flow Qs
of 0.350 microLiters per minute, then the fluidic resistance,
Rs=Ps/Qs, of about 25,700 psi/.mu.L/min would be calculated and
stored in memory. The instrument 11 would use the following formula
to determine the new settings:
Pnew=Qnew*Rs
[0109] In order to achieve the new settings, the control means 125
commands the linear valve 127 to increase pressure in the conduits
to the operating pressure as shown in ramp 161a and to the
operating flow as shown in ramp 161b. At the same time, the control
means 125 runs the pumps 115a, 115b under closed loop feedback
control. By setting the pumps 115a, 115b to a high pressure and
allowing the flow to increase to meet the pressure for a breif
interval, the set flow and pressure parameters are acheived very
quickly without ruining the integrity of the mixture. In one
embodiment, step S7 lasts about 30 seconds or preferably less.
[0110] The ramps 141a, 141b, 145a, 145b, 149a, 149b, 161a and 161b
are depicted as being linear; however, such ramps may be non-linear
as long as the slope, indicative of the change in pressure over
time is managed. A preferred slope is not greater than
approximately 10,000 pounds per square inch (psi) per second. More
preferably, the slope is in the range of 10 to 1,000 psi per
second, and, more preferably, 100 to 500 psi per second.
[0111] At step S8, once the instrument 111 reaches the desired
setting for flow and pressure, the flow enters a steady state
condition during the elution run as shown by flat area 163b. In
contrast, the pressure may fluctuate as shown by varying region
163a. During step S8, the sample is delivered to the analytical
column 167 for analysis.
Feed Forward Embodiments
[0112] Refering now to FIGS. 4A-10, another HPLC instrument 211
using feed forward is referred to and shown in various views. As
will be appreciated by those of ordinary skill in the pertinent
art, the instrument 211 utilizes similar principles to the
instrument 111 described above. Accordingly, like reference
numerals preceded by the numeral "2" instead of the numeral "1",
are used to indicate like elements whenever possible. A primary
difference of the instrument 211 in comparison to the instrument
111 is the lack of a linear valve 127.
[0113] Referring to FIGS. 4A and 4B, another direct-flow nano-scale
HPLC instrument 211 that uses open-loop feed forward compensation
in accordance with the subject technology is shown. The instrument
211 is shown in somewhat schematic form with a plurality of parts
grouped and collectively referred to as a binary gradient pump 220.
The binary gradient pump 220 is designed for use in
analytical-scale chromatography and modified to deliver precise
gradients for nano-flow LC applications by incorporating in-line
flow transducers 233A, 233B, used in conjunction with closed-loop
feed back control provided by the control means 225. In one
embodiment, the flow transducers 233A, 233B are anemometer type
flow transducers.
[0114] Pump 215A represents an independent high-pressure pump that
sources one of two mobile-phase solvents (e.g., aqueous) from a
reservoir supply 235A to the mixing tee 223. Pump 235A may be
comprised of two reciprocating pumps for smooth flow delivery. The
solvent flow at the outlet of pump 235A is directed through an
inline pressure transducer 231A and an inline nano-flow transducer
233A. The pressure transducer 231A provides a measurement of the
instrument operating pressure and the flow transducer 233A provides
direct nano-flow measurement of the solvent A fluid stream into the
mixing tee 223. Through closed-loop control, the control means 225
is able to maintain accurate flow delivery in the presence of large
parasitic flow leakages upstream from the flow transducer 233A, due
to high-pressure seals and check valves of pump 215A. Likewise,
pump 215B represents a similar independent high-pressure pump
having a corresponding pressure transducer 231B and flow transducer
233B that sources the complementary mobile-phase solvent (e.g.,
organic) from a reservoir supply 235B to the mixing tee 223.
[0115] The control means 225 establishes the desired user-set bulk
flow to the instrument 211 and compositional mix ratio of the two
solvents by regulating the delivery flow of each pump 215A, 215B
into the mixing tee 223. By having both pressure and flow
transducers 231A, 231B, 233A, 233B in each pump's solvent stream,
the instrument 211 provides great flexibility with two fundamental
control modes of operation.
[0116] Conduits 240A, 240B are series restrictors such as, for
example, short lengths of capillary tubing, to provide passive
fluidic decoupling between the two pumps 215A, 215B to stabilize
the inherent interactions of the two flow control loops across the
mixing tee 223. The restrictors or conduits 240A, 240B are
intentionally located downstream of the flow transducers 233A, 233B
to minimize volume between the transducers 233A, 233B and the tee
223.
[0117] The solvent stream from the binary gradient pump 220 is
conveyed via a conduit (e.g., a short capillary tube) to a sample
injector valve 219 that introduces one or more sample analytes from
a sample reservoir into the fluid stream, which is directed to one
or more separation columns 265, 267, depending on the
configuration. The separated analytes from the analytical column
267 are directed to a detector 269 such as a mass spectrometer, UV
detector and/or the like.
[0118] As shown in FIG. 4B, the instrument 211 features a
simplified trapping scheme, operating in a forward flush mode,
comprising a trapping column 265, a trapping valve 221, and an
analytical column 267. The binary gradient pump 220 performs both
operations of sample loading/trapping and gradient delivery as
described below. While FIG. 4B indicates a "forward flush" scheme,
the subject technology is applicable to other trapping approaches
such as "reverse flush" as is known to those familiar with the art
of trapping schemes. During trapping operation as shown, the binary
gradient pump 220 provides a fixed solvent composition (e.g., very
high % A (aqueous) very low % B organic) at a relatively high flow
rate (e.g., 5 to 15 uL/min) with the trapping valve 221 opened to
waste. As a result of the very high fluidic restriction of the
analytical column 267, the solvent stream is directed to waste. The
sample analytes are loaded onto the trapping column 265 while the
eluent components that are undesirable or harmful to the analytical
column 267 (e.g., buffers, salts, and the like) are flushed away to
waste. When trapping completes, the binary gradient pump 220 stops
the trapping flow and reconfigures the instrument 211 for the
analytical separation. The trapping valve 221 closes, thus
directing the fluid stream to the analytical column 267. The binary
gradient pump 220 then commences with the gradient run at the
preprogrammed nano-flow rate and time-programmed solvent
composition profile. As noted in FIG. 4A, the "Set Flow",
percentage of solvent A and percentage of solvent B are entered by
a user into the control means 225.
[0119] In another embodiment, the instrument 211 operates in direct
injection mode without the trapping column 265. In that mode, a
short capillary tube (not shown) replaces the trapping column 265,
and the trapping valve 221 is kept closed, directing the fluid
stream to the analytical column 267. The injection of the sample
analytes takes place during the start of the gradient run.
[0120] Referring now to FIG. 5, a somewhat schematic representation
of the control means 225 in greater detail is shown to illustrate a
flow control strategy. As noted above, prior art systems suffered
from the loss of solvent compositional control during operation at
very high system pressures, such as greater than 5,000 psi, and
between the operations of trapping or direct injection runs
whenever the flow to the system started or stopped. The control
means 225 establishes the user-set bulk flow to the instrument 211
(Set Flow) and the user-set compositional solvent ratios (% A and %
B) through two flow controllers 250A, 250B for each solvent pump
235A, 235B, respectively. The set flow, percentage of solvent A and
percentage of solvent B are input to each path, respectively, along
summing junctions 254A, 254B.
[0121] Each flow controller 250A, 250B is implemented as a PID feed
back is controller and behaves essentially as a servo loop,
responding to either an instrument flow change or a solvent
composition change, either of which is conveyed as a new flow set
point. The flow controllers 250A, 250B compare the current flow set
point or reference input (Qra, Qrb) to the measured flow (Qa, Qb)
entering the tee 223 and, in the case of stepper motor controlled
piston pumps, produce the appropriate motor velocity signal, which
drives the necessary pump volumetric flow rate (Qpa, Qpb) into the
instrument 211 so that the reference flow (Qr) is satisfied.
Similarly, for other pumping strategies, appropriate signals Qpa,
Qpb would be generated to satisfy Qr.
[0122] Set-flow changes are conveyed to the flow controllers 250A,
250B through a linear ramping function, represented by the ramp
means 252A, 252B. For each pump 215A, 215B, the ramp means 252A,
252B passes the new flow set point to the reference set point (Qr)
of the respective flow controller 250A, 250B incrementally as a
linear function with respect to time over a ramping interval Tr
that assures good servo tracking between the two flow controllers
250A, 250B. Generally, a reasonable value for Tr is about five
times the time constants of the flow transducers 233A, 233B. In a
preferred instrument 211, the time constant of the flow transducers
233A, 233B is about 5 seconds, so a ramping time interval Tr of 30
seconds is preferred. The ramp means 252A, 252B maintains the prior
history of the respective flow controller's set point and for each
new user-set value, the control means 225 calculates the following:
the direction; the total change value delta Flow (equal to
newSetFlow-presentSetFlow); and the ramp rate Rp (equal to delta
Flow/Tr), which governs the incremental updates to the reference
input (Qr) of the flow controller 250A, 250B.
[0123] As can be seen from FIG. 5, each ramp means 252, flow
controller 250, transducer 231, 233 and pump 215 form a flow
control servo loop. For relatively small changes in composition
(i.e., complementary flow changes between the two flow control
servo loops) or bulk flow changes to the instrument 211 (i.e.,
changes within the band width of the flow transducers), the two
flow control servo loops are able to maintain good composition
control, hence flow tracking, sufficient to do HPLC chromatography.
Such is the case during the programmed gradient run.
[0124] However, when making very large flow changes, accompanied
with large back pressure changes, the tracking ability of the two
flow controller servo loops breaks down, and composition control is
lost, resulting in composition error in the fluid stream of the
instrument 211 and a loss of chromatography. For example, when the
flow in the instrument 211 is stopped or started with very
restrictive capillary columns, composition error can occur. This
problem is particularly problematic during trapping when
transitioning the bulk flow of the instrument 211 from the end of
the trapping phase (e.g., high-flow and moderate pressure) to the
starting analytical flow conditions (e.g., nano flow and very high
pressure) at which gradient delivery begins.
[0125] The ramp means 252A, 252B alone can limit large set point
changes to the flow controllers reasonably effectively with lower
operating pressures, such as an operating pressure of 5,000 psi.
However, if not properly compensated for, a higher operating
pressure, such as 10,000 psi, to enable operating with much longer
capillary columns, yields at least a partial loss of composition
control, due to severe mistracking of the flow controllers.
[0126] The cause of the mistracking is due to the very large
cylinder volumes of the pumps 215A, 215B relative to the nano-scale
of flow delivery to the instrument columns 265, 267. Since the
mobile phase solvents used in these LC applications are relatively
compressible at the aforementioned operating pressures, the captive
volume of these fluids requires considerable energy storage before
steady-state flow can be achieved into the mixing tee 223. This
energy storage manifests itself as a compressible volume change of
the instrument fluid and is directly proportional to the solvent
compressibility and captive volume, and proportional to the square
of the operating pressure. The stored energy is supplied by each
pump 215 as an additional `charging` flow while the pressure is
changing and is much greater than the flow being controlled into
the mixing tee 223. Because the solvents A, B typically have much
different fluid compressibility, the effective charging flows
between the two respective pumps 215A, 215B differ by the ratio of
their compressibility as does the volume changes across the mixing
tee 223. Thus, the inability of the flow controllers 250A, 250B to
manage the considerable imbalance of charging flows and volumes at
the mixing tee 223 creates a loss of composition control.
Fluidics Model
[0127] To illustrate the dynamics of the loss of composition
control and how the instrument compensates for the limitations of
the flow controllers 250A, 250B, a simplified analytical model of
the fluidics is now presented. The compressibility of a fluid is
defined as the relative volume change of a fluid as a response to a
pressure change and is expressed mathematically as:
.beta. = - 1 V .differential. V .differential. P ##EQU00001##
where V is the volume of the system, and the partial derivative
expresses the change in volume to the change in pressure. In
thermodynamic terms, the compressibility is defined by the
conditions or process by which the partial differential is taken
(e.g., adiabatic/constant entropy or isothermal/constant
temperature).
[0128] The differential change in volume relative to the change in
pressure is also expressed as fluid capacitance, which is the
capacity of a fluid to store energy. Thus, the fluidic capacitance
becomes:
C=.beta.V.sub.o
where .beta. is the fluidic compressibility and Vo is the captive
volume of the system. Using the simple Ohmic relation from
electrical circuits, the fluidic resistance is represented as:
R=.DELTA.P/Q
where .DELTA.P is the pressure drop across a restrictive element,
and Q is the flow rate through the element. Resistance R is
basically proportional to the length of a fluidic element and the
viscosity of the fluid flowing therethrough.
[0129] Using the basic lumped-parameter elements of capacitance and
resistance analogous to electrical circuits, an equivalent fluidic
circuit of the instrument 211 is presented in FIG. 6. In the
analogy, currents "I" represent volumetric flow "Q" in units of
.mu.L/min. Voltages represent pressures in psi and capacitance "C"
represents the fluidic energy storage of the fluids and is
expressed in units of .mu.L/psi. Resistance "R" represents fluidic
restriction to flow in units of psi/.mu.L/min.
[0130] Relating the fluidic model in FIG. 6 to the flow control
servo loops in FIG. 5, Ipa and Ipb are two dependent current or
flow sources that represent the volumetric flow delivered by both
pumps A and B into the instrument 211, respectively. The flows Ipa,
Ipb are directly proportional to the commanded velocity of the pump
motor actuators. PTa and PTb represent the two in-line pressure
transducers 231A, 231B, respectively. The pressure transducers
231A, 231B measure the node pressures Pa and Pb, respectively, at
the pumps 215A, 215B. Likewise, FTa and FTb represent the two
in-line flow transducers 233A, 233B, respectively. The flow
transducers 233A, 233B measure the two controlled flows Ia and Ib,
respectively, into the mixing tee 223.
[0131] The sum of the two flows Ia and Ib at the mixing tee 223
results in the flow Is out of the mixing tee 223. The effective
load of the instrument 211 is represented by resistance Rs, which
is primarily the fluidic resistance of the columns 265, 267 at the
end of the fluidic circuit, where Cs represents a lumped fluidic
capacitance of the instrument downstream of the mixing tee 223. The
capacitance Cs is primarily comprised of the volume of the sample
loop of the injector valve 219 and the volume of the trapping
column 265. The capacitance Cs is much smaller than the pump
capacitances Ca, Cb.
[0132] Ra and Rb represent the fluidic resistance of the two
capillary restrictors 240A, 240B, respectively. While not
necessary, the resistances Ra, Rb are made roughly equal by making
the respective lengths in proportion to the viscosities of solvents
A, B. The resistances Ra, Rb are very small relative to the
combined resistance Rs of the capillary columns 265, 267. For
example, the resistances Ra, Rb are about 40 psi/.mu.L/min, whereas
the resistance Rs of a 75 .mu.m ID.times.150 mm.times.1.7 .mu.m
particle capillary column is approximately 25,000 psi/.mu.L/min for
water.
[0133] Ps is the system back pressure resulting from the system
flow Is into the columns 265, 267 or instrument resistance Rs.
Since the capillary restrictors are so much smaller than Rs, each
of the measured pump pressures Pa, Pb are a reasonable measure of
the instrument back pressure Ps. Ca and Cb represent the lumped
fluidic capacitance of each pump 215A, 215B, respectively, and
comprises the captive volume of: the final delivering pump
cylinder, including its displacement stroke volume and internal
dead volume between its inlet valve (or check valve, which is not
shown) and outlet port, the pressure transducer, and all the
connecting conduits up to but not inclusive of the flow transducers
231, 233, respectively. In one embodiment, the captive volume of
the delivering pump cylinder and interconnecting conduits up to the
flow transducers is about 160 uL. For the aqueous pump 215A, the
fluid compressibility constant .beta. for water is 3.12e-6/psi.
Thus the fluid capacitance of the A pump is about:
Ca=3.12e-6/psi*160 .mu.L=0.0005 .mu.L/psi
For the organic pump 215B, the fluid compressibility constant
.beta. for acetonitrile (ACN) is 7.34e-6/psi, which is 2.35 times
as compressible as water. Thus, the fluid capacitance of the B pump
is about:
Cb=7.34e-6/psi*160 .mu.L=0.00118 .mu.L/psi
[0134] Ica and Icb represent the compressibility `charging` or
`discharging` flows into or out of the respective pump fluidic
capacitances Ca, Cb whenever the node pressures Pa, Pb change.
Using the electrical equivalency, the compressibility flows are
expressed as follows:
I ca = C a .times. d .times. P a d .times. t .times. .times. I c
.times. b = C b .times. d .times. P b d .times. t ##EQU00002##
To put the limitations of the feedback control strategy in
perspective, consider the following scenario in which the flow
ramps from zero to the initial conditions of the gradient over the
ramping interval Tr of 30 seconds. Such is the case either
following trapping or with direct injection. During the ramping
interval, the composition is held constant with a very high aqueous
to organic mix ratio (e.g., 97% A, 3% B). For a target flow rate Is
of 350 nL/min with the aforementioned column, a steady-state system
pressure Ps of about 9,000 psi is to be achieved. With the ramping
scheme, the instrument pressure Ps should also ramp linearly from
zero to 9,000 psi. The change in fluid volume for the A pump needed
to compress the system to 9,000 psi is:
.DELTA.V.sub.a=C.sub.a.DELTA.P=0.0005 .mu.L/psi*9,000 psi=4.5
.mu.L
Since the compressibility of the B solvent is 2.35 times that of
the A solvent, the corresponding volume change of the B pump is
about 10.6 .mu.L.
[0135] For example, iIn order to ramp the system pressure to 9,000
psi over the 30 second ramp interval, the pump 215A must supply a
sustained average `charging` flow Ica of:
I ca = C a .times. .DELTA. .times. P a .DELTA. .times. T = 0 . 0
.times. 005 .times. .times. L .times. / .times. psi * 9 , 000
.times. .times. psi / 30 .times. .times. sec = 9.0 .times. .times.
L .times. / .times. min ##EQU00003##
while ramping an associated controlled flow Ia from zero to a
target value of 0.97*350 nL/min or about 340 nL/min. Likewise, the
pump 215B must supply a sustained average `charging` flow Icb
of:
I c .times. b = C b .times. .DELTA. .times. P b .DELTA. .times. T =
0 . 0 .times. 0118 .times. .times. L .times. / .times. psi * 9 ,
000 .times. .times. psi / 30 .times. .times. sec = 21.2 .times.
.times. L .times. / .times. min ##EQU00004##
while ramping an associated controlled flow Ib from zero to a
target value of 0.03*350 nL/min=11 nL/min.
[0136] If composition control at the outlet of the tee is to be
maintained, then the compression or charging flows at the pump
heads must be maintained at the correct ratio of the two solvent
compressibility factors, independent of the composition ratios. If
this requirement is not met, then the delicate differential
tracking of the node pressures Pa, Pb across the inlet ports (not
shown) of the mixing tee 223 necessary to maintain the proper mix
ratio of the controlled inlet flows Ia, Ib will no longer be
maintained by the flow controllers 250A, 250B. If the differential
pressure upset is severe enough, then one of the node pressures Pa,
Pb will drop below the instrument pressure Ps causing a flow
reversal for that leg or side of the mixing tee 223. In this
situation, the dominating pump with the higher node pressure, Pa or
Pb, saturates the solvent mixture at the outlet (not shown) of the
mixing tee 223 and contaminates the other pump's flow transducer,
233A or 233B as the case may be. Thus, due to the structure and
volume scale of the fluidic circuit, the pressure differential
across the mixing tee is much more strongly dependent on the
charging flows as compared with the two controlled flows into the
tee. This poses a very ill-conditioned problem for the feed back
controllers, which can only react to errors in the rather small
inlet flows into the mixing tee compared with the much larger
uncontrolled charging flows, which are in the order of almost 2,000
to 1 for the organic pump.
[0137] For example, at the onset of the ramping operation, there
will be virtually no measurable flows into the mixing tee 223 until
the capacitance of each pump head begins to charge. The flow
controller's proportional action would rail the command signal
velocity of each pump motor (not shown) to the high limit of 100
uL/min, which is about 10 times the average charging flow of the
aqueous pump 215A, but barely 5 times the average charging flow of
the organic pump 215B. Thus, the time duration in which both pumps
215A, 215B remain open loop is different, being about 2.7 seconds
for the aqueous pump 215A, and about 6.3 seconds for the organic
pump 215B. Clearly, there is an overlapping interval of at least 3
seconds in which both pumps 215A, 2I5B are open loop but flowing at
the same flow rate! Thus, the feed back controllers have no
knowledge or direct measurements of the very high charging flows
caused by the discharged state of both pump heads and are unable to
maintain composition control. This problem has a complimentary
behavior when the system flow is ramped to zero from the typical
ending gradient conditions. In this case, the stored energy of each
pump 215A, 215B is released back into the system 211 when the
compressed volumes expand. Since the organic pump 215B has more
capacitance or energy storage, the fluid of the aqeous pump 215B
expands more than the fluid of the organic pump 215A, causing the
organic pump 215B to undesirably `burp` out into the outlet stream
and back flow into the other transducer 233A. Such is the case when
the system 211 transitions from the previous run to begin either a
direct injection or a trapping operation for the next run.
[0138] Referring to FIG. 7, a somewhat more detailed version of the
instrument 311 of FIGS. 4A and 4B is shown to more particularly
illustrate the usage of feed forward compensation in accordance
with the subject technology. As will be appreciated by those of
ordinary skill in the pertinent art, the instrument 311 utilizes
similar principles to the instruments described above. Accordingly,
like reference numerals preceded by the numeral "3" are used to
indicate like elements whenever possible. A primary addition of the
instrument 311 in comparison to the instruments 111, 211 is the
inclusion of signal summing and multiplying junctions and analogous
current references as depicted in the fluidics model of FIG. 6.
[0139] In overview, the corrective feed forward control scheme is
based on separation of the bulk energy storage, i.e., the bulk
fluid compressibility of the pump heads from the feed back control
of flow (and composition) during the flow ramping operation. The
feed-forward scheme manages the large back pressure changes
required to compress and decompress the bulk captive head volumes
independently from the closed-loop flow controllers 250A, 250B. The
feed forward scheme applies a control law to the tandem movement of
the pump plungers (not shown) such that the storage or release of
compression energy of the pump heads (not shown) are controlled in
such a manner to preserve the proper compositional flows, Qa and
Qb, at the input ports to the mixing tee 23. The feed forward
control action effectively balances the two node pressures, Pa and
Pb, so that the sloshing of liquid across the mixing tee 323 is
within acceptable error tolerance of composition.
[0140] From the perspective of the feed back controllers, the feed
forward scheme off loads the bulk capacitive load or disturbance to
the flow controllers during the flow ramps, effectively making the
captive volume of the pump heads virtually disappear. A primary
load disturbance to the controlled flows is delta P, or change in
node pressure from the driving pump head. Thus, the disturbance
flow Qdist presented to each flow controller is proportional to
delta P. This delta P gives rise or fall to the very large
compression or decompression flow Ic that flows into and out of the
captive head volume whenever large pressure changes occur due to
the large bulk flow changes. The feed forward loop effectively
estimates the necessary charge or discharge flow Ic that must be
supplied or absorbed by each pump 315A, 315B, independently from
the controlled flows into the mixing tee 323, so that the feedback
controllers can maintain the correct compositional mix ratio while
the system pressure ramps in response to only the change in bulk
flow to the system 311. Referring back to FIG. 6, generally, feed
forward control law is simply the charging or discharging flow of
the head volume capacitance in response to a change in
pressure:
Qfeedforward=Ic=C dp/dt
and the pressure change is the estimated or measured disturbance
function.
[0141] Referring now to FIG. 7, the ramp means 352 provides the
desired bulk flow ramping function to the control strategy. For the
A pump 315A, using a measure of the system resistance Rs stored
from a previous run by the control means, an estimate of the
anticipated system pressure ramp Pa is derived at multiplier 254A1.
The feed forward scheme computes the pressure disturbance signal by
taking the derivative operation 255 of Pa. The feed forward control
law Qca is calculated by multiplying the derivative of Pa by the
measured capacitance Ca of pump A at 259. The feed forward
correction is summed into the command signal from the flow feed
back controller 350A at summing junction 263. Likewise, the B pump
315B derives its feed forward correction signal via operations,
254A4, 257, 261, and 265. In practical applications, the feed
forward calculation need not take the actual derivative of the
pressure signal, with the control scheme presented in FIG. 7.
Instead, the ramp rate Rp, computed by the ramp means 352 (cite
paragraph 0123), is the derivative of the pressure ramp signal and
is simply multiplied by the capacitance Ca to produce the feed
forward signal Qca.
[0142] In another embodiment, the feed forward estimate of the
disturbance to signal Pa could replaced by an actual measurement
provided by pressure transducer 331A. However, noise is always
present with the signal and adequate means of filtering must be
provided appropriate to taking the derivative of a signal.
[0143] Referring now back to FIG. 7, a somewhat more detailed
version of the instrument of FIGS. 4A and 4B is shown to more
particularly illustrate the application of feed forward
compensation in accordance with the subject technology.
Further Refinements of Feed Forward
[0144] FIG. 8 is another more detailed version of an LC instrument
shown to more particularly illustrate particular components of a
refined feed forward signal in accordance with the subject
technology. FIG. 9 is another electrical circuit analog to an LC
instrument of having a system or load flow Is to provide energy or
charge Cs to the load in accordance with FIG. 8. For simplicity,
FIG. 8 depicts only the fluid stream or leg of the pump 415A into
the mixing tee 423, and all explanations of the fluid refinements
described here also apply symmetrically across the mixing tee 423
to the leg of pump 415B.
[0145] A first refinement of the feed-forward control involves
consideration of the changing volume of the pump 415A and resultant
capacitance Ca during the delivery stroke. As the stroke traverses
from bottom dead center to top dead center of the pump 415A, the
effective pump capacitance Ca varies with a net change proportional
to the ratio of the stroke volume to the sum of the stroke and
fixed captive volumes. Since the stroke volume can be significant,
Ca is a time-varying parameter, as shown in FIG. 9, and can be
corrected during feed-forward control. A proper correction is made
by monitoring the plunger travel position and making the
appropriate change to the varying captive volume Vo before
multiplying by the solvent compressibility factor .beta.. The
plunger position is known by reading either the direct commanded
motor position or by measuring a motor shaft encoder, linear
position indicator and the like as provided. The correction to the
time-varying value of Ca is depicted in FIG. 8 as stroke volume
correction to Ca before multiplier 459.
[0146] A second refinement of the feed-forward control scheme
involves consideration of the captive fluid volume of the in-line
flow transducers and the related undesirable energy storage
effects. In one embodiment, each flow transducer 433A, 433B is
constructed from a cylindrical tube (not shown) with a captive
volume of just over 2 .mu.L. The measuring elements are arranged
symmetrically along the length of the tube, roughly splitting the
captive volume evenly upstream and downstream from a mid point,
which is the virtual node at which the flow measurement is made.
The energy storage effects caused by these captive volume elements
can be represented as two shunt capacitances, CpreFT and CpostFT in
FIG. 8, flanking the transducer 433A.
[0147] FIG. 9 incorporates the upstream and downstream capacitances
CpreFT and CpostFT that flank the flow transducer. In FIG. 9, FTa
represents an ideal virtual transducer that produces a measured
flow signal at a vanishingly small point centered between the
flanking capacitances of the physical device. The transducer
capacitances CpreFT and Cpost give rise to the corresponding shunt
currents, Icpr and Icpo, whenever the pump 415A pressure at node Pa
changes. The refinements made to the feed-forward control scheme
account for these additional shut currents as shown in FIG. 9
entering multipliers 471, 473 and passing into summing junctions
475, 477. These feed-forward flows compensate the transducer
charging currents needed to satisfy the commanded pressure ramp at
the output of multiplier 454A1. For practical purposes, the
upstream transducer capacitance, CpreFT, is subsumed by the much
larger pump cylinder volume or capacitance Ca, so the effect of the
shunt current Icpr on the measured flow is negligible. Thus, the
flow through the transducer substantially consists of the flow of
the pump 415A Iat into the mixing tee 423 and the charging flow
Icpo, due to the down-stream capacitance of the flow transducer
433A. In practical applications, the capacitance CpostFT that gives
rise to the charging flow lcpo accounts for any captive volume of
the series restrictor 440A, which can be minimized by using
capillary tubing. With this model, the flow transducer 433A
produces a measurement error of the desired control flow, Iat,
whenever the pressure Pa of the pump 415A changes. The error term
is the post transducer charging flow Icpo.
[0148] A third refinement is made to the control scheme by
correcting the flow transducer measured flow error at the feedback
controller 450A as shown in FIG. 8 by summing junction 479, which
sums the feed-forward calculated value Icpo into the setpoint of
the flow controller 450A. To see how the correction is
accomplished, the flow controller 450A responds to an internal
error detector signal, set point minus measurement, which is
expressed as (Qra'-Qa). The set point term Qra' is the actual
driving set point Qra and the computed error term of the flow
measurement Icpo. However, the flow transducer measurement responds
to the sum of lat and Icpo. Thus, the flow error term is cancelled
across the flow controller's error signal, producing the to desired
error signal (Qra-Qat).
[0149] Referring to FIG. 10, still another more detailed version of
an LC instrument shown to more particularly illustrate a hybrid
approach using a feed forward signal in accordance with the subject
technology. The control scheme shown in FIG. 10 can also be
referred to as a hybrid pressure and flow control strategy or
hybrid scheme. The hybrid scheme addresses the compressibility
elements downstream of the mixing tee 523. In one embodiment, the
downstream elements comprise user configurable components, making
characterization by the control strategy impractical if possible at
all. Such components include the injector sample loop and trapping
columns, which are offered in various volume sizes. These
components represent a lumped system capacitance attached to the
outlet tee 523 and are represented in the fluidic models of FIGS. 6
and 8 as Cs, which appears in parallel with the very restrictive
system resistance Rs of the analytical column (not shown). As noted
above, using feed forward compensation can solve the composition
control problems up to the mixing tee 523 that plague a
feedback-only control strategy. However, when a large-volume
component such as a sample loop and/or trapping column is added
downstream of the mixing tee 523, the downstream capacitance Cs
presents a very large charging flow demand to one or both of the
flow controllers, depending on the mix ratio, while the flow ramps
are in progress. Due to the magnitude of Cs, the system charging
flow is much greater than the ramped bulk flow to the system and
can easily exceed the flow range capability of the flow transducers
533A, 533B. The extra flow of Cs forces the flow transducers 533A,
533B to go open loop, resulting in a loss of composition
control.
[0150] With the hybrid scheme, the control strategy for one of the
flow legs into the mixing tee 523 is changed from flow control to
pressure control, for example the leg of the pump 515A. The other
leg, having the pump 515B, operates in flow control mode, with feed
forward correction and all the refinements described earlier. In
practice, the choice leg to use under flow control is the one
handling the smallest contribution to the bulk flow, which is
typically the organic pump 515B. Such is the state of the flow when
starting and stopping the gradient or analytical run. During flow
ramping with the hybrid scheme, the organic pump 515B under flow
control with feed-forward compensation of compressibility,
maintains the required stiffness under essentially a zero-flow
condition to prevent sloshing of liquid across the mixing tee 523.
Meanwhile, due to the complimentary nature of the two pump flows
into the mixing tee 523, the aqueous pump 515A, under pressure
control, supplies all the necessary charging flows, both up steam
of the flow transducer 533A, and the charging flows downstream of
the mixing tee 523. The complimentary pressure and flow mode of the
hybrid approach prevents the feed back controller 550B of the
flow-controlled leg from going open loop, while the
pressure-controlled leg supplies the energy down stream of the
mixing tee 523 needed to compress the rest of the system to follow
the bulk flow ramp. With pressure mode incorporated into the
control scheme, the flow-controlled leg of the pump 515B is
prevented from going open loop and is able to keep up with its
contribution to the bulk flow of the system, thus maintaining
correct composition control into the system. In another embodiment,
the hybrid scheme can incorporate the feed-forward correction
elements 459, 463, 471, 473, 475, 477 shown in FIG. 9 into the
output command signal of the pressure controller leg of the pump
515A of FIG. 10 to further provide enhanced error prevention and
correction.
Performing Gradient Embodiments
[0151] Referring to FIG. 11, another direct-flow nano-scale HPLC
instrument 611 in accordance with the subject technology is shown.
As will be appreciated by those of ordinary skill in the pertinent
art, the instrument 611 utilizes similar principles to the
instruments described above. Accordingly, like reference numerals
preceded by the numeral "6" are used to indicate like elements
whenever possible. A primary difference of the instrument 611 in
comparison to the instruments above is the use of a storage
capillary 622 to retain a preformed gradient. As would be
appreciated by those of ordinary skill in the art, the subject
methods and systems described herein can be used together,
separately and in any combination to achieve the desired
performance. Similar to above, the HPLC instrument 611 has two
major componets, a binary solvent gradient delivery subsystem 620
and an analytical subsystem 604. The solvent delivery subsystem 620
forms and provides a gradient to the analytical subsystem 604,
which generates a chromatogram thereon. It is envisioned that the
solvent delivery susbsystem 620 could be adapted to many
alternative analytical designs to provide the advantages of the
subject technology. In breif overview, the solvent delivery
subsystem 620 performs relatively low pressure mixing of solvents A
and B in one step to form a gradient and subsequent high pressure
delivery of the gradient to the analytical subsystem 604 in a
second step. These steps are orthogonal in that the first mixing
step is independent and does not interfere with the second delivery
step. The solvent delivery subsystem 620 is binary in that two
pumps 615A, 615B are employed. Each pump 615A, 615B produces an
output directed through an inline pressure transducer 631A, 631B
and a flow transducer 633A, to 633B, respectively. A controller 625
governs the operation of the pumps 615A, 615B and receives signals
from the transducers 631A, 631B, 633A, 633B.
[0152] A mixing node or tee 623 combines the pump outputs to form
the desired solvent mixture, which flows into a storage capillary
622 to form a gradient. Preferably, the mixing tee 623 is a
T-shaped fitting and the storage capillary 622 is sized to minimize
backpressure and dispersion. Downflow from the storage capillary
622, a nano-tee fitting 624 forms two outlets. One outlet of the
nano-tee fitting 624 connects to the analytical subsystem 604 while
the other outlet connects to a dump valve 626. The operation of the
dump valve 626 is also governed by the controller 625 such that
during forming the gradient in the storage capillary 622, the dump
valve 626 is open to direct resident fluid to waste while both
pumps 615A, 615B run. During delivery of the gradient to the
analytical subsystem 604, only pump 615A runs and the other pump
615B is "offline". Preferably, the selection and arrangement of the
components of the instrument 611 moves the outlet of the solvent
delivery subsystem 620 from the outlet of the mixing tee 623 to the
outlet of the nano-tee fitting 624. The dump valve 626 is
preferably a pin valve. But instead of a pin valve, you could use a
ball valve, a gate valve, a globe valve, a butterfly valve and the
like.
[0153] The analytical subsystem 604 includes a transport line 628,
such as a short capillary tube, that connects the outlet of the
solvent delivery subsystem 620 to a sample injector 619. The sample
injector 619 introduces one or more analyte samples, stored in
sample retainer 642, into the gradient or fluid stream. Once the
analyte is present, the fluid stream is directed to a column 667
for analysis. In the column 667, the analytes are isolated,
separated and directed to a detector 669. Preferably, the detector
669 is any type, such as a mass spectrometer, an ultra-violet
detector and the like, that is suitable for the particular
application. Based upon the readings of the detector 669, a
chromatogram is generated. Preferably, pump 615A is an independent
high-pressure pump that sources one of two mobile-phase solvents
(e.g., an aqueous solvent) from a reservoir supply (not shown) to
the mixing tee 623. Similar to above, the solvent flow at the
outlet of the aqueous pump 615A is read by the inline pressure
transducer 631A to provide a measurement of the instrument
operating pressure. The flow transducer 633A provides a direct
nano-flow measurement of the aqueous solvent upon entry into the
mixing tee 623. Each of these signals is passed to the controller
625 for closed-loop control of the aqueous pump 615A. Thus, the
controller 625 is able to maintain accurate flow delivery upstream
from the flow transducer 633A despite the presence of large
parasitic flow leakages due to high-pressure seals, check valves of
the aqueous pump 615A and similar issues well-known to those of
ordinary skill in the pertinent art.
[0154] Similarly, pump 615B is another independent high-pressure
pump feeding the pressure transducer 63IB and flow transducer 633B.
However, in contrast, pump 615B sources a complementary
mobile-phase solvent (e.g., an organic solvent) from a reservoir
supply (not shown) to the mixing tee 623. Because the signals of
the pressure transducer 631B and flow transducer 633B are also fed
to the controller 625, complete closed-loop control is possible.
The controller 625 establishes a desired user-set bulk flow to the
analytical subsystem 604 as well as the compositional mix ratio of
the two solvents by regulating the delivery flow of each pump 615A,
615B into the mixing tee 623. In one embodiment, the conduits from
the flow transducers 633A, 633B are capillary restrictors to
provide passive fluidic decoupling between the two pumps 615A, 615B
to stabilize the inherent interactions of the two flow control
loops across the mixing tee 623. In another embodiment, the
capillary restrictors are provided upstream from the flow
transducers 633A, 633B. In either case or when both are used,
feedback instability from the two pumps 615A, 615B is avoided as
well as cross-flow and/or back-flow contamination.
[0155] Gradient Formation and Delivery
[0156] Referring now to FIG. 12, a process for operating the
solvent delivery subsystem 620 to provide a gradient to the
analytical subsystem 604 is shown as a flow diagram. At step S1,
prior to gradient formation, the controller 625 readies the solvent
delivery subsystem 620 by opening the dump valve 626 to direct the
old resident fluid (e.g., 100% aqueous solvent) inside the storage
capillary 622 to waste. It is envisioned that any suitable conduit,
tube or microfluidic structure now know or later developed could be
used to perform the function of the storage capillary 622.
Formation of the gradient with the storage capillary 622
essentially vented to atmosphere accomplishes three functions: 1)
the formation back pressure is accurately controlled by the
geometry of the storage capillary 622, independent of the column
667 or other connected consumables; 2) the fluid in the storage
capillary 622 is purged to waste to prevent upsetting the
equilibrium state of the column 667 between injection runs; and 3)
any leading or trailing compositional aberrations bracketing the
formed gradient, due to starting and stopping the flow during
formation, are directed away from the primary fluid stream of the
instrument 611, i.e., away from the analytical subsystem 604 and
the column 667 therein. At step S2, the gradient is formed in the
storage capillary 622 by operating the pumps 615A, 615B at low
pressure (e.g., 100 psi) and higher flow rate (e.g., ten to twenty
times the normal chromatographic flow rate). Preferably, the
storage capillary 622 receives the gradient in a FIFO fashion. Once
the gradient is formed in the storage capillary 622, delivery to
the analytical subsystem 604 can occur later, at high pressure and
at the normal chromatographic flow rate. The geometry of the
storage capillary 622 is sized by length and inner diameter to
achieve the necessary storage volume capacity for the gradient and
to minimize the formation of backpressure and gradient dispersion.
In addition to the entire gradient, the volume capacity of the
storage capillary 622 is preferably large enough to accommodate the
additional overhead of transport volume necessary to move the
gradient from the storage capillary 22 to the column 667.
[0157] Still referring to FIG. 12, at step S3, the organic pump
615B is taken offline to ready for delivering the gradient. Thus,
the organic pump 615B does not participate in gradient delivery to
the analytical subsystem 604. In a preferred embodiment, the
organic pump 615B is taken offline by the controller 625 by
maintaining closed-loop flow control of the organic pump 615B with
a reference flow setting of zero. In another embodiment, the
organic pump 615B is taken offline by employing an isolation valve
(not shown) upstream from and adjacent to the mixing tee 623.
[0158] At step S4, the controller 625 closes the dump valve 626 to
direct flow of the formed gradient to the analytical subsystem 604.
As a result, the aqueous pump 615A, operating under closed-loop
flow control, is solely used as the prime mover to push the
gradient out of the storage capillary 622 for delivery at step
S5.
[0159] Flow Control For Multiple Columns
[0160] For applications that require changing the operational flow
rate for a particular choice of column during an injection run,
e.g., sample trapping and 2-D chromatography, the flow rate is
started and stopped between selection of each column. The solvent
delivery subsystem 620 of FIG. 11 is well-suited to provide varying
gradients in such LC systems despite relatively large cylinder
volumes of the pumps 615A, 615B and the nano-scale of flow delivery
to the columns. In one embodiment, the controller 625 resets the
pumps 615A, 615B with the dump valve 626 open to waste and waits
for the solvent delivery subsystem 620 to achieve steady state
flow. Although effective, this operation can be undesirably time
consuming because of the relatively large volume change of stored
energy required to compress the fluid stream up to the to
operational back pressure.
[0161] For example, the compressibility constant (i.e., change in
volume per change in pressure) of the aqueous pump 615A is
preferably about 0.5 nL/psi. The time required to reach a
steady-state pressure of about 9,000 psi at a flow rate of 300
nL/min to a particular capillary column [e.g., 75 .mu.m
ID.times.250 mm.times.1.7 .mu.m particles] would be about 15
minutes as shown in the calculation below.
.DELTA.V=C.DELTA.P=0.0005 uL/psi*9,000 psi=4.5 uL
T=.DELTA.V/q=4.5 uL/0.300 uL/min=15 min
[0162] Referring now to FIG. 13, a process is shown that avoids a
slow flow startup when performing a series of injection runs by a
solvent delivery subsystem to a LC system (not shown) having
multiple columns. Although not shown separately, this solvent
delivery subsystem can be exactly as represented in FIG. 11. Thus
for clarity, the reference to the solvent delivery subsystem 620 of
FIG. 11 is again utilized. In breif overview, the solvent delivery
subsystem 620 commences flow delivery under pressure control to
rapidly compress the system pressure to a former steady state flow
condition from the previous run, then transitions to flow
control.
[0163] The process of FIG. 13 commences from the end of an
injection run when the column is to be re-equilibrated to the
starting conditions for the next run. At step S1 of FIG. 13, the
controller 625 receives and stores a flow rate selected by a user
for the next column to be used. At step S2, the controller 625
stores the system pressure measurements from the pressure
transducers 633 as was desired for the previous column injection.
At step S3, the flow is stopped, and a new gradient is formed, as
described above with respect to FIG. 2. To commence flow delivery,
the controller 625 configures the aqueous pump 615A to operate
under closed-loop pressure control, using the pressure transducer
633A as feedback.
[0164] At step S4, the controller 625 sets the reference set point
(e.g., the target pressure) to the system pressure measurement
stored from the previous run. Then, the aqueous pump 615A is run to
attain the reference set point. Once the system pressure reaches
the reference set point, i.e., compresses back to the steady state
pressure, the process of FIG. 13 proceeds to step S5.
[0165] At step S5, the controller 625 transitions the aqueous pump
615A back to closed-loop flow control, using the flow transducer
633A as feedback, and sets the reference set point to the user-set
elution flow rate received at step S1. Upon reaching the desired
elution flow rate, the process proceeds to step S6 in which
delivery of the new gradient occurs. Preferably, the aqueous pump
615A, operating under closed-loop flow control, is solely used as
the prime mover to push the gradient out of the storage capillary
622 for delivery.
[0166] Further variations to the design shown in FIG. 11 are also
well within the scope of the subject technology. For example,
without limitation, the storage capillary 622, the nano-tee fitting
624, and the dump valve 626 are shown before the injector 619 in
FIG. 11. To minimize the gradient transport delay, these elements
622, 624, 626 could be located as close as possible to the injector
619. As a result, the transport line or element 628 can be
eliminated and replaced by the storage capillary 622. Moving the
storage capillary 622 in closer proximity to the sample injector
619 and column 667 also presents the opportunity to co-locate the
storage capillary 622 with the column 667, which is often placed in
a thermally-managed compartment. Such placement of the storage
capillary 622 favorably isolates the storage capillary 622 from
external temperature changes, enhancing retention-time
reproducibility.
[0167] For another example, the subject technology can readily be
adapted to WATERS.RTM. nanoACQUITY UPLC.TM. System, avalilable from
Waters Corporation of Milford, Mass., with the addition of a
storage capillary and a dumping valve. In these instruments, the
operational pressure is extended from 10,000 to a pressure rating
of 15,000 psi or beyond, while the operational delivery flow rate
is capable of being about 10 nL/min. Further, the WATERS.RTM.
nanoACQUITY UPLC.TM. System can provide the variable flow/peak
parking feature for enhanced sensitivity as discussed in more
detail below and the feed forward compensation discussed above.
Trapping Embodiments
[0168] The subject technology is also advantageously applied to
trapping applications that use short trapping columns (i.e., high
captive volume applications) in series with very restrictive
columns. In such circumstances, the stopping of flow is
characterized by a long time interval in which the pressure finally
subsides to near zero. The long time interval is due to the very
large effective time constant of the trapping system created by the
highly capacitive trapping column (i.e., the stored energy of
compression) and the large restriction of the analytical column
(i.e., the limited flow). The effective time constant can be many
tens of minutes. To overcome this long time constant in trapping
applications, the principles of the subject solvent delivery
subsystems can be utilized. Further, by efficiently forming the
gradient, usage of and wear upon the typical trapping components,
such as valves, can be minimized to extend the life thereof.
[0169] Referring now to FIG. 14, a solvent delivery system 720 is
shown in use in a trapping application system 711. As will be
appreciated by those of ordinary skill in the pertinent art, the
system 711 utilizes a similar solvent delivery subsystem 720 as
shown in FIG. 11. Accordingly, like reference numerals preceded by
the numeral "7" are used to indicate like elements whenever
possible. In the system 711, the operations of sample loading,
sample trapping and gradient formation are preferably
serialized.
[0170] The system 711 includes a sample manager 720 connected to a
heating trapping module 704. The sample manager 720 includes an
inject or load valve 742 connected to the solvent delivery
subsystem 720 and the heating trapping module 750. A syringe 744
and needle 746 are also connected to the inject valve 742. The
heating trapping module 750 has a trap column 752 in series with an
analytical column 767, the trap column 752 being connected to the
inject valve 742. A tee fitting 756, intermediate the trap column
752 and analytical colum 767, connects to a trap valve 758.
[0171] For clarity, it is noted that pressure transducers are not
shown in FIG. 14. The pressure transducers are present as an
integral component of the pumps 715A, 715B. However, a small
restrictor 770 is shown connected immediately upstream from the
flow transducer 733A and a large restrictor 771 is shown connected
immediately upstream from the flow transducer 733B, whereas such
restrictors are preferably present but omitted in other embodiments
herein. The restrictors 770, 771 serve to attenuate the potential
interaction between the pumps 715A, 715B because of the mutual
connection to the mixing tee 723. In effect, the restrictors 770,
771 minimize the backflow between the pumps 715A, 715B across the
mixing tee 723. As the pump 715A acts as the primary mover and
moves the most viscous liquid (e.g., water), the associated
restrictor 770 is relatively smaller. In a preferred embodiment,
the small restrictor 770 is approximately 0.025 cm inner
diameter.times.20 cm long and the large restrictor 771 is
approximately 0.025 cm inner diameter.times.45 cm long. In another
preferred embodiment, the small restrictor 770 is approximately 25
.mu.m I.D. by 20 cm and the large restrictor 771 is approximately
25 .mu.m I.D. by 150 cm. While the restrictors may be different,
the restrictors may also have the same value and dimensions.
[0172] To load a sample, the needle 746 is placed in the sample in
a well-known manner. The syringe 744 provides suction to draw the
sample through the needle 746 into the inject valve 742, which is
set so that the flow passes from point 3 to point 4 to point 1 to
point 2 of the inject valve 742 to the needle 746. Upon completion,
the sample is located in a loop 745 between points 4 and 1 of the
inject valve 742. At this point, the sample is ready to be cleaned
and concentrated by being put onto the trap column 752.
[0173] During sample trapping, exact control of the flow is not
required but high flow is desirable in order to move the sample
from the loop 745 to the trap column 752. Typically, the sample is
capillary scale such as approximately 4 uL/min. In a preferred
embodiment, the solvent delivery system 720 moves 5-15 uL/min for a
period of 5 minutes to move the sample onto the trap column 752 at
only 1,000 psi.
[0174] For sample trapping, the conditions of the heating trapping
module 750 advantageously transition from high pressure/low flow to
low pressure/high flow. To accomplish this, the solvent delivery
subsystem 720 stops flow by rapidly decompressing the heating
trapping module 750 under pressure-control mode. The controller
(not shown) accomplishes the rapid decompression by reconfiguring
the aqueous or driving pump 715A from flow control to pressure
control mode, using the internal pressure transducer as feedback.
Then, the controller sets the reference pressure set point to zero
and commences operation of the pump 715A. Preferably, the organic
pump 715B is similarly controlled. In another embodiment, the
aqueous pump 715A alone runs during sample trapping.
[0175] Still referring to FIG. 14, when the system 711 runs pumps
715A, 715B, the pin valve 726 is closed to direct flow into the
inject valve 742 at point 5. The inject valve 742 is set so flow
passes from point 5 to point 4 to point 1 to exit at point 2
carrying the sample therewith. The flow then passes into the
heating trapping module 750 with a trap valve 758 being open to
waste at point 6. As a result, the flow is directed through the
trap column 752 but thereafter passes through the tee fitting 756
to waste via point 1 to point 6 of the trap valve 758. As a result
of this flow, the sample passes onto the trap column 752.
[0176] Next, the system 711 forms the gradient to be used in the
elution run. The solvent delivery subsystem 720 is run as described
above to form the gradient. In particular, the pin valve 726 is
open to waste and the pumps 715A, 715B are run until the gradient
is in the storage capillary 722. Preferably, this results in a
system pressure between ambient and 300 psi but high flow.
[0177] To perform an elution run, the solvent delivery subsystem
720 has pump 715A run as described above to deliver the gradient.
The gradient enters point 5 of the inject valve 742 and exits point
6 into the trap column 752. With the trap valve 758 effectively
closing the path to waste, the flow passes through the tee fitting
756 and analytical column 767 to the mass spectroscopy system (not
shown). Even though the organic pump 715B is not delivering flow
during the elution run, the pump 715B also compresses while set to
zero flow. Otherwise, the pump 715A would force flow across the
mixing tee 723 resulting in contamination. Alternatively, the
contamination could be allowed and, prior to subsequent gradient
formation, the pumps 715A, 715B could be run to waste for
cleansing.
[0178] Referring now to FIG. 15, another solvent delivery system
820 is shown in use in a trapping application system 811. As will
be appreciated by those of ordinary skill in the pertinent art, the
system 811 utilizes similar principles to the systems described
above. Accordingly, like reference numerals with the first number
of "8" are used to indicate like elements whenever possible. A
primary difference of the system 811 is the addition of an
auxiliary solvent manager (ASM) 860 and modified plumbing to
accommodate the ASM 860. The system 811 allows for more parallelism
between the operations of sample loading, sample trapping and
gradient formation. Additionally, less flow interruption and a
single composition trapping operation can be realized.
[0179] The ASM 860 has an additional pump 862 connected to point 5
of the inject valve 842. Rather than connecting to the trap column
852, the point 6 of the inject valve 842 connects to point 4 of the
trap valve 858. The output of the solvent delivery subsystem 820
connects to point 2 of the trap valve 858 such that the trap column
852 can receive flow from the inject valve 842 or solvent delivery
subsystem 820 as selected by the controller (not shown).
[0180] It is envisioned that the sample loading and the gradient
formation can be performed simultaneously. By having the output of
the solvent delivery subsystem 820 connected to the trap valve 858,
the sample manager 440B is isolated such that these operations can
occur simultaneously. Additionally, sample trapping and gradient
formation can occur in parallel. While the gradient if formed as
described above with respect to FIG. 14, the pump 862 pushes flow
into the inject valve 842 at point 5 to point 4, through the loop
845 through point 1 to exit at point 6. The flow continues into the
trap valve 858 at point 4 to exit point 3 into the trap column 852.
Upon exiting the trap column 852, the flow moves to waste via tee
856 and again into the trap valve 858 at point 1 to exit at point
6.
[0181] During an elution run, the system 811 operates very
similarly to that described above with the aqueous pump 815A being
the primary mover of fluid while the organic pump 815B compresses
but is set to zero flow. Consequently, the gradient moves from the
storage capillary 822 into the trap valve 858 at point 3 to exit at
point 2. From the trap valve 858, the flow passes through the trap
column 852 into the analytical column 867 because the path to waste
is closed.
[0182] The configuration of system 811 also advantageously allows
for component optimization. For example, pump 862 typically runs at
high flow, say 20 uL/min whereas the aqueous pump 815A may run at a
fraction of a uL/min. Rather than trying to use a single transducer
to cover this flow range, each pump 815A, 862 can have a flow
transducer specifically tuned for the relevant range.
Peak Parking Embodiments
[0183] Referring now to FIG. 16, a process is shown that rapidly
reduces the elution flow rate of the solvent delivery subsystem 620
to the analytical subsystem 604 of, for example, FIG. 11. FIG. 11
is referred to again for clarity and as the same components can be
used to efficiently accomplish this peak parking. It is envisioned
that the peak parking can also be performed with the configuration
of many other fluidic chromatography systems, such as that shown in
FIG. 4. In FIG. 11, only the delivery pump, typically 631A, would
"ramp" to the new desired flow since that is the only pump
responsible for fluid delivery when a gradient is being delivered
to the analytical column 676. In the case of FIG. 4, both
transducers 231AB, 233AB would be utlized to control the ramping
procedure. At step S1 of FIG. 16, upon detection of a single
elution peak of interest by the detector 669, the controller 625
receives a signal to "park" or reduce the bulk flow to the
analytical subsystem 604. In one embodiment, the analytical
subsystem 604 sends the park signal to the controller 625. At step
S2, the controller 625 stores the existing system pressure
measurement from the pressure transducers 631A, 631B.
[0184] At step S3, the controller 625 calculates a new reduced
target pressure and switches from closed-loop flow control to
closed-loop pressure control using the pressure transducers 631A,
631B..sup.2 Preferably, the controller 625 calculates the new
reduced target pressure by assuming a linear `Ohmic` fluidic load
of the LC instrument 611 according to the following formula
R=delivery pressure divided by delivery flow
where R is a restrictive load. In other words, to determine the
reduced pressure, the controller 625 targets the reduced pressure
needed by the same ratio as the desired flow rate reduction. For
example, consider a system delivering fluid at 300 nL/min at a
system pressure of 9000 psi. This would correlate to a system load
of 30 psi/(nL/min). If the target flow rate was 50 nL/min, then the
target pressure for this system would be 1500 psi.
[0185] Depending on the duration of the peak park event, the
controller 625 may transition back to flow control mode at the
reduced flow setting, for more accurate and reproducible flow
delivery. Operating in pressure mode gives the advantage of a
faster decompression response time, since the flow transducers
633A, 633B have much less signal bandwidth compared to the pressure
transducers 631A, 631B. As a result, the elution flow rate can be
reduced by up to fifty times or even more.
[0186] At step S4, detection of the elution peak is completed and
the controller 625 restores the flow back to the normal elution
flow rate by reversing steps S1-S3, using the stored system
pressure measurement as the new pressure target. The advantages of
this approach over conventional high-pressure mixing schemes is
that the problems associated with corruption of the gradient when
compressibility energy is taken out or put back into the fluid
stream are eliminated because the gradient is preformed and only a
single fluid driving pump (e.g., the aqueous pump 615A) is used to
affect the flow change.
Co-Locating a Gradient Storage Device with Column Embodiments
[0187] Referring now to FIGS. 17-19, performance of an LC
instrument can be further improved by reducing gradient delay time,
i.e., the time taken to delivery the gradient, whether or not
preformed, from the storage capillary or location to the column. In
brief overview, the gradient delay time can be reduced by
co-locating or directly connecting the gradient storage device to
the column. Further, gradient dispersion is also reduced by the
co-location and sample injection time is reduced by using a higher
gradient forming flow rate for injection.
[0188] Referring to FIG. 17, a somewhat schematic block diagram
illustrating a LC instrument using a gradient storage device
without a vent valve is referred to generally by the 911. As will
be appreciated by those of ordinary skill in the pertinent art, the
LC instrument 911 utilizes similar principles to the systems
described above. Accordingly, the following discussion is largely
directed to the differences. Similar to the systems above, the LC
instrument 911 includes an injection valve 942 with a loop 945 for
delivering a gradient to the analytical column 967 of a MS detector
969. The injection valve 942 is connected to a binary pump 920,
syringe 944 and sample container 946.
[0189] A difference of the LC instrument 911 is that a gradient
storage device 922 is provided intermediate the injection valve 942
and the analytical column 967. The gradient storage device 922
allows gradient loading at a high flow rate and delivery at a
normal flow rate. The backpressure generated when the gradient is
loaded can be many times higher than that created when the gradient
is run. For example, with an analytical column 967 of 25 um inner
diameter by 10 cm in length, packed with 3.5 .mu.m particles,
sample loading and gradient pre-formation occurs at 250 nl/min
under about 8,000 psi of pressure with the analytical column 967
online to reduce sample loading time. Whereas, gradient delivery
occurs at 25 nl/min under about 800 psi of pressure for ultra high
sensitivity analysis. Because the gradient is loaded at a high flow
rate, the sample can be injected at the same time to effectively
reduce runtime (e.g., the sample loading time portion of the
runtime).
[0190] The gradient storage device 922 may be directly connected to
the analytical column 967. By having the gradient storage device
922 closely located to the analytical coumn 967, the delay in
deliverying the gradient is minimized and dispersion of the
gradient is reduced. The gradient storage device 922 may be as
simple as an empty section of capillary tubing with or without any
filling matrix. The inner diameter of the gradient storage device
922 may be a similar diamater to that of the analytical column 967
but the volume of the gradient storage device 922 is preferably
equal or somewhat greater than the gradient volume. In another
embodiment, the gradient storage device 922 is integral with the
analytical column 967.
[0191] To further shorten the gradient loading time, the total
gradient delay volume (i.e., the injection loop volume and the
gradient storage device volume) is made equal or slightly greater
than the gradient volume. In view of the above advantages, it is
envisioned that the subject technology has wide application. For
example, it could be advantageously applied in high throughput
separations such as the second dimension separation of an offline
2D system or in ultra-sensitive analysis using narrower
nano-columns (e.g., 25 um i.d.) to form a gradient at about 300
nl/min and deliver the gradient at about 30 nl/min.
[0192] Referring to FIG. 18, a somewhat schematic block diagram
illustrating another LC instrument using a gradient storage device
with a vent valve is referred to generally by the reference number
911A. To denote that the LC instrument 911A has many of the same
components of the instrument 911, the suffix "A" is used to
identify like elements whenever possible. The LC instrument 911A is
similar to that above but includes a trap column 952A and a vent
valve 941A intermediate the gradient storage device 922A and the
analytical column 967A. By using a vent valve 941A, the LC
instrument 911A is well-suited for use with a trap column 952A. The
addition of the vent valve 941A allows operating the LC instrument
911A at a loading pressure lower than the running pressure if
desired.
[0193] Referring now to FIG. 19, still another LC instrument 911B
is shown with another arrangement for allowing operating the LC
instrument 911B at a relatively low loading pressure. To denote
that the LC instrument 911A has many of the same components of the
instruments 911, 911A, the suffix "B" is used to identify like
elements whenever possible. The LC instrument 911B includes a
T-connection 923 intermediate the trap column 952B and the
analytical column 967B. The T-connection 923 extends to a binary
pump 920B which can ultimately create a flowpath to waste as shown
and described above. In effect, the T-connection 923 performs the
same function as the vent valve 941A of FIG. 18. The LC instrument
911B also has an isocratic pump 962 and a trap valve 958 similar to
that shown and described above with respect to FIG. 15. As can be
seen from these co-location examples, it is also envisioned that
the sample is injected simultaneously with loading the gradient
using a very high flow rate to reduce the sample loading time.
[0194] As would be appreciated by those of ordinary skill in the
pertinent art, the subject technology is applicable to use not only
as a solvent delivery subsystem in a variety of applications with
significant advantages for low flow, high pressure applications but
could be advantageously used in many applications. For example, the
subject technology is very applicable to systems without a trapping
column. The functions of several elements may, in alternative
embodiments, be carried out by fewer elements, or a single element.
Similarly, in some embodiments, any functional element may perform
fewer, or different, operations than those described with respect
to the illustrated embodiments. Also, functional elements (e.g.,
pressure and flow transducers and the like) shown as distinct for
purposes of illustration may be incorporated within other
functional elements, separated in different hardware or distributed
in various ways for a particular implementation. Further, relative
size and location are merely somewhat schematic and it is
understood that not only the same but many other embodiments could
have varying depictions.
[0195] While the invention has been described with respect to
certain illustrative embodiments, those skilled in the art will
readily appreciate that various changes and/or modifications can be
made to the invention without departing from the spirit or scope of
the invention.
INCORPORATION BY REFERENCE
[0196] The contents of all references (including literature
references, issued patents, published patent applications, and
co-pending patent applications) cited throughout this application
are hereby expressly incorporated herein in their entireties by
reference.
EQUIVALENTS
[0197] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *