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WO2001088528A2 - Systemes chromatographiques a haut rendement - Google Patents

Systemes chromatographiques a haut rendement Download PDF

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Publication number
WO2001088528A2
WO2001088528A2 PCT/US2001/015141 US0115141W WO0188528A2 WO 2001088528 A2 WO2001088528 A2 WO 2001088528A2 US 0115141 W US0115141 W US 0115141W WO 0188528 A2 WO0188528 A2 WO 0188528A2
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WO
WIPO (PCT)
Prior art keywords
chromatographic
compounds
mobile phase
eluent
detector
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PCT/US2001/015141
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English (en)
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WO2001088528A3 (fr
Inventor
Charles Pidgeon
Nadege Rooke
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Admetric Biochem Inc.
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Priority to CA002408375A priority Critical patent/CA2408375A1/fr
Priority to AU2001261392A priority patent/AU2001261392A1/en
Publication of WO2001088528A2 publication Critical patent/WO2001088528A2/fr
Publication of WO2001088528A3 publication Critical patent/WO2001088528A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N2030/628—Multiplexing, i.e. several columns sharing a single detector
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8804—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 automated systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G01N30/46—Flow patterns using more than one column
    • G01N30/466—Flow patterns using more than one column with separation columns in parallel
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/84—Preparation of the fraction to be distributed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8651—Recording, data aquisition, archiving and storage
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8658—Optimising operation parameters

Definitions

  • This invention relates to the use of a chromatographic system for comparing physicochemical properties of compounds in a compound mixture. More particularly, the present invention is directed to a gradient flow chromatographic system for measuring and comparing numerical values characteristic of the interaction of compounds in a test mixture solution with a solid phase substrate.
  • the gradient flow system offers advantages for acquiring and comparing data in connection with chemometric analysis and in high throughput drug screening protocols where the data is used alone or in combination with other molecular descriptors for prediction of biological activity.
  • the present invention relates to a method for efficient computer implemented determination of not only chemical structure, but also the physicochemical properties, derived from chromatographic data, critical for such QSAR studies and drug discovery efforts. More particularly, the present invention provides a method of choice for creating databases containing algorithm accessible data derived from chromatographic analysis of complex mixtures needed for such QSAR studies.
  • chromatographic systems in such drug discovery methodology has limitations.
  • the compound components both control compounds and test compounds, can have widely distributed retention times (t r ), ranging from fractions of a minute to hours. While the retention times can be reduced by using mobile phase gradients of increasing or decreasing polarity (depending on the nature of the solid phase), changes in mobile phase composition can result in unpredictable variability in calculated k' values. Such variability can be attributed to the differences in thermodynamic equilibrium properties arising from varying the composition of the mobile phase. Accordingly, researchers have focused their attention on optimizing chromatographic throughput efficiency without resorting to use of mobile phase gradient elution protocols and at the same time minimizing need for additional capital expenditures.
  • the eluent switch allows cost-effective use of costly eluent analyzers (detectors), such as a mass spectrograph, by incorporating a simple and relatively inexpensive switching device immediately upstream (eluent flow-wise) from the eluent analyzer.
  • Solvent programming i.e., variation of the mobile phase composition as a function of time
  • Solvent programming is widely used in chromatography as a way to speed up elution time, mainly because it is so easy to achieve and control.
  • the scope of its application is limited due to the fact that changing the composition of the mobile phase affects a lot more than just its polarity/eluotropic strength.
  • changing the ratio buffer/organic modifier affects the degree of ionization of the buffer species (i.e., the ionic strength of the solution) and thus the pH of the solution.
  • chromatographic surfaces that contain ionizable functional groups will be sensitive to pH changes and/or ionic strength of the mobile phase.
  • solvent composition gradients may change the local pKa at the interface between mobile phase and stationary phase.
  • thermodynamics of the mobile phase/surface interactions, and the chromatographic properties of the surface will be modified.
  • degree of ionization will change with mobile phase composition, and so will their pKa.
  • this method cannot be employed for the determination of physicochemical descriptors characteristic of the solute/surface thermodynamic interactions or solute partitioning between stationary and mobile phases, such as the capacity factor (k'). This observation has been reported by Scott et al. (Scott R. P., Lawrence J.
  • Temperature programming is not often used in chromatography, perhaps because the effects of temperature changes on separation are complex, involving changes in both solute retention and band broadening. Nearly all the physical parameters that play a role in LC separation are a function of temperature. Changing temperature strongly affects mobile phase viscosity and the degree of ionization of buffer solutions. It has been also shown that temperature variation may induce stationary phase conformation transition, and the observation of this physicochemical phenomenon has initiated the development of temperature- responsive stationary phases as an additional tool for chromatographic selectivity optimization. Most of all, temperature variation affects the thermodynamics of solute/stationary phase interactions. Therefore, temperature programming is restricted to applications where the purpose of the chromatographic run is not the collection of physicochemical parameters or other descriptors of the dynamics of the binding/interaction of the solutes with the chromatographic surface.
  • Flow rate programming as a means to speed up separation time in liquid chromatography is seldom used and has found limited applications in HPLC. Studies involving flow gradient elution have been reported, but they remain scarce and limited to very specific cases. The potential of gradient flow elution in chromatography has been largely overlooked. Flow programming has not been used as a general method for accelerating data acquisition, minimizing instrument time, or for the collection of chromatographic databases (large numbers of chromatographic parameters characteristic of physical, chemical or biological properties of the compounds studied).
  • test compounds can be calculated, or they can be determined empirically with use of, for example, liposomes, immobilized artificial membranes, such as those described in U.S. Patent No. 4,931 ,498, Langmuir Blodget films, computer chips or similar devices with immobilized lipids, capillary zone electrophoresis columns coated with membrane lipids, and the like.
  • immobilized artificial membranes IAMs
  • the numerical values characteristic of membrane affinity are determined chromatographically using an aqueous mobile phase and a stationary phase comprising a membrane mimetic surface as defined in U.S. patent 4,931,498.
  • Membrane binding properties of a set of test compounds of unknown biological activities are compared to the membrane binding properties of control compounds having known in vivo biological activity to assess the probability that the test compounds will exhibit one or more biological activities in vivo.
  • the ordered set of numerical values for each control compound or each set of control compounds can be represented by the expression ⁇ C l5 C 2 , ..., C > wherein n is the number of membrane mimetic surfaces identified and used in the screening method.
  • a similar ordered set of numerical values ⁇ T j , T 2 , ..., T n > for each test compound characteristic of its biological relevant interaction with each of the respective membrane mimetic surfaces is determined.
  • the set of numerical values for each test compound is then compared with the set of respective values for the control compounds, and the biological properties of those control compounds having ordered sets of numerical values best matching the respective numerical values in the ordered set of values for the test compound are identified.
  • Pattern matching using vector calculus, multivariate analysis or principal component analysis of the numerical values characteristic of the test compounds and the control compounds allows comparison of the membrane binding properties of the test compounds and each of the control compounds or, if the control compounds all have a common biological activity/property, average or mean membrane binding values of the set of control compounds for each membrane mimetic surface.
  • Such drug discovery protocols are described in detail in PCT International Publication No. WO 99/10522.
  • the highlighted compounds are the compounds for which a switch in elution order occurs.
  • changes in elution order occur three times. This implies that the elution time or capacity factor at different fractions of acetonitrile is not affected to the same extent for each compound.
  • This has a significant effect on data acquisition for creating chromatographic databases, particularly when the data to be incorporated in the database are membrane affinity fingerprints (MAFs).
  • MAFs membrane affinity fingerprints
  • the MAF of a compound is defined as an ordered set of n numerical values (a vector) characteristic of the affinity of the compound for n membrane mimetic surfaces or Immobilized Artificial Membranes (IAMs). See WO 99/10522 for a detailed description.
  • Figs. 2a-b illustrates how a change in elution order affects the classification/comparison of compounds in membrane space.
  • compounds A and B are characterized by vectors [1, 3, 2] and [2, 1, 2] respectively (Graph 2a), in the three-dimensional membrane space (coordinate system) defined by IAM1, IAM2, and IAM3 (the numerical values defining the vectors reflect the elution time or affinity of the compounds for each surface: higher values mean higher retention affinity).
  • compounds A and B were eluted on the same surfaces with a different mobile phase composition, such that the elution of A and B on IAM 2 is reversed: A elutes before B (for simplicity considerations, let us assume that the retention of both compounds is not significantly changed on IAM 1 and IAM 3).
  • the MAF vectors of compounds A and B are now [1, 2, 2] and [2, 3, 2], respectively.
  • the change in elution order has dramatically altered the position of each compound in membrane space, as shown in Figs. 2a-b.
  • the process of membrane affinity fingerprinting (MAFing) is based on the comparison of MAF vectors in membrane space. As illustrated in Figs. 2a-b, a change in elution order causes compounds A and B to be defined by radically different vectors, which is unacceptable for MAFing purposes, because it may lead to misclassification.
  • chromatographic database The value of a chromatographic database is reflected not only by the nature/type of information that it contains, but also by the range of compound diversity that it encompasses. Collecting data for creating meaningful and robust chromatographic databases implies that compounds be eluted faster: (1) to minimize data collection time and (2) to include strongly retained compounds. People routinely resort to solvent gradients to reduce compounds elution times. However, as extensively discussed above, this chromatographic technique implies non-linear dependence of retention volumes or capacity factors of the solutes with mobile phase composition, and a change in chromatographic behavior of the solutes, which is compound specific.
  • a suitable solution to this problem would be to find chromatographic conditions that would allow the use of constant mobile phase composition (isocratic conditions) for all the compounds comprising the database, regardless of their relative affinities for the chromatographic surfaces to be used.
  • the retention volume of any given compound is independent of the flow rate used to perfuse the column (for any given mobile phase composition, the volume of mobile phase required to elute a compound of a column is a constant, and does not depend on the flow rate).
  • compounds may elute from columns after very small or very large volumes of mobile phase have been perfused through the columns.
  • compound mixtures may contain compounds with very low and very high affinity for a chromatographic surface.
  • the early eluting compounds will easily be detected and characterized, whereas the late eluting peaks will likely be so broad that they will be difficult to detect by any method. Furthermore, at 4.0 mL/min the early eluting peaks elute so quickly that the peak shapes can not be characterized and that retention times are difficult to measure, but the late eluting compounds are easily characterized. Thus getting a complete set of chromatographic data for the compound mixture C1-C10 would require two chromatographic runs: one at 0.5 mL/min and one at 4.0 mL/min.
  • a better approach yet is to collect the data with a flow gradient, ramped or stepped from 0.5 mL/min to 4.0 mL/min.
  • Chromatographic data for both early and late eluting compounds could be obtained with acceptable peak shape characterization and peak detection, all of which being accomplished in one single run and in a minimum amount of time.
  • the present invention focuses on the use of flow gradients for establishing chromatographic databases (e.g., the collection of large numbers of chromatographic data characteristic of chemical, physical, biological and/or thermodynamic properties of solutes) for mixtures of chemically diverse compounds exhibiting a broad range of affinities for the chromatographic surface in use.
  • the primary advantage of the present invention is that it provides a solution to a problem routinely encountered in the field of chromatography: the non-linear dependence of solute retention time with mobile phase composition (in particular organic modifier concentration).
  • This phenomenon due in part to a change in the thermodynamics of solute/stationary phase interactions, makes the use of solvent gradients (modification of the mobile phase composition as a function of time) or organic modifier extrapolation techniques unsuitable for the characterization of said thermodynamic interactions for mixtures of compounds with radically different chromatographic affmities (i.e., poorly retained compounds versus solutes with high retention).
  • This problem has high significance for disciplines such as chemometrics, determination of physicochemical properties, prediction of biological properties, and drug discovery in general.
  • the flow gradient method described herein allows the efficient, reliable, and accurate determination of chromatographic parameters (characteristic of solute thermodynamic properties) for a wide variety of structurally, physicochemically, and biologically diverse compounds. Thus, it is particularly suitable for the collection of large databases containing empirical data derived from chromatographic analyses.
  • the present invention provides a chromatographic method that allows faster elution of higher retained solutes.
  • a chromatographic system allowing highly efficient determination of physicochemical properties and/or chemical structure identification, through the use of a chromatographic unit (capable of delivering an accurate flow gradient according to a pre-set profile) in communication with one eluent analyzer, is described.
  • Such a system dramatically increases the performance, efficiency, scope of use and commercial value of said chromatographic unit/eluent analyzer system.
  • the chromatographic unit can be any chromatographic system that can be interfaced with an analyzer or detector, and can include (but is not limited to) high-performance liquid chromatography (HPLC) columns, capillary electrophoresis chromatography (CEC) columns, Gas Chromatography (GC) columns, super-critical fluid columns and microchips.
  • HPLC high-performance liquid chromatography
  • CEC capillary electrophoresis chromatography
  • GC Gas Chromatography
  • the eluent analyzer unit is any instrument capable of identifying the presence, physicochemical characteristics and/or chemical structure of a compound, including (but not limited to) a mass spectrometer (MS), a Fourier transform infra red spectrometer (FTIR), a Fourier transform ultra violet spectrometer (FTUV), standard UV detector, fluorescent detector, electrochemical detector, and a Fourier transform nuclear magnetic resonance spectrometer (FTNMR).
  • MS mass spectrometer
  • FTIR Fourier transform infra red spectrometer
  • FTUV Fourier transform ultra violet spectrometer
  • standard UV detector fluorescent detector
  • electrochemical detector electrochemical detector
  • FPNMR Fourier transform nuclear magnetic resonance spectrometer
  • the present design permits faster collection of the HPLC profiles (including structural identification) of a mixture of multiple compounds eluting from one chromatographic unit.
  • the implementation of the method described therein allows a more efficient and cost-effective use of a costly eluent analyzer unit (such as
  • the chromatographic process represents a reversible equilibrium of solutes between a mobile phase and the stationary phase in a chromatographic system.
  • the magnitude of the solute retention is a direct result of this equilibrium, and it is typically expressed by a parameter known as the capacity factor, k'.
  • k' is equal to (t R -t 0 )/t 0 where t 0 is the dead time of the chromatographic unit and t R is the retention time of the respective solutes.
  • the capacity factor can be expressed as a function of N 0 , the so-called dead volume of the chromatographic unit, and N R the retention volume, i.e., the volume of mobile phase required to elute the respective solute from the chromatographic unit, e.g., an HPLC column.
  • the capacity factor is therefore a numerical value characteristic of a mass distribution equilibrium of the solutes between the mobile phase and the stationary phase in a chromatographic unit, and its determination allows the calculation of various physicochemical values according to pre-determined algorithms.
  • a linear program rate is often used, but the method is not limited to this flow rate profile.
  • the various flow rate programs that may be used in chromatography can be classified as ramp, step and multi- segment.
  • the initial flow rate, the final flow rate, and the programming rate define the theoretical program (equation).
  • the retention volume of a specific solute is a constant value at any flow rate. Knowledge of the mathematical expression describing the variation of flow rate as a function of time allows the determination of the retention volume of any compound at any given time.
  • the retention volume of a specific solute under gradient flow conditions can be calculated by integrating the time profile of the employed flow rate programming pattern.
  • the present invention derives from a continuing effort to enhance throughput in chromatography-based drug discovery/chemometric methodologies. It is based on use of a gradient flow elution protocol for the expedient determination of k' values and other numerical values characteristic of the interaction of test compounds and control compounds with a solid phase substrate.
  • the present chromatographic system and method eliminates the need to vary mobile phase component ratios and the consequent interference with the determination of numerical values characteristic of the interaction of the test and control compounds (i.e., the solutes and the mobile phase), with the surface of the stationary phase.
  • the improved performance of the present system derives from use of variable elution flow rate chromatographic system capable of measuring and recording the retention volume (N R ) for each solute eluted from the chromatographic unit.
  • a chromatographic system for comparing and or determining physicochemical properties of compounds in a sample including a mixture of compounds, particularly represented as numerical valves characteristic of the interaction of the compounds with the stationary phases in the chromatographic system.
  • the mixture can include test compounds and one or more compounds of known biological activity.
  • the chromatographic system comprises a chromatographic unit having a sample loading port, a mobile phase delivery port, an eluent exit port, and a stationary phase.
  • the stationary phase can be any commercially available stationary phase detailed for use in chromatographic applications.
  • Preferred stationary phases include the membrane mimetic surfaces described in U.S. Patent No. 4,931 ,498, and, for example, immobilized macromolecules.
  • the improved chromatographic system of the present invention further comprises a mobile phase supply system including a pump (mechanical, osmotic, etc.) and a source of mobile phase for delivering the mobile phase to the mobile phase delivery port of the chromatographic unit.
  • the chromatographic unit is in the form of a column containing a solid phase of the sort designed for use in classical HPLC systems.
  • the present chromatographic system also includes a detector in fluid flow communication with the eluent exit port.
  • the detector has an eluent sampling port and is capable of providing a signal of the presence or identity of a compound in eluent delivered to the detector sampling port.
  • the detector is capable of providing a signal characteristic of the detected solute compound.
  • the detector can comprise a mass spectrometer, a Fourier transform infra-red spectrometer, a Fourier transform ultraviolet spectrometer, or a Fourier transform nuclear magnetic resonance spectrometer.
  • the detector comprises a mass spectrometer (MS).
  • MS mass spectrometer
  • the MS detector provides a signal of total ion concentration in aliquots of eluent delivered to the sampling port of the detector.
  • Another element of the present chromatographic system is a controller for the pump component of the mobile phase supply system for controlling the flow rate of mobile phase through the chromatographic unit at a programmed flow rate.
  • the controller is also designed to provide signals indicative of the volume of mobile phase delivered to the chromatographic unit after delivery of the sample to the unit.
  • the chromatographic system includes a flow rate reporter and a timer for providing signals indicating the time following delivery of a sample to the sample delivery port. Signals from the timer and the flow rate reporter can be directed to a microprocessor programmed to calculate the volume of mobile phase delivered to the chromatographic unit at any point in time.
  • the controller for the pump is programmed to modulate the rate of flow of mobile phase through the chromatographic unit according to a predetermined flow rate profile.
  • the controller can be programmed to adjust the flow rate in a stepped profile.
  • the chromatographic system of the present invention further comprises a data management system in communication with the pump controller and the detector.
  • the data management system comprises a data storage unit for storing signals from the detector as a function of volume of mobile phase delivered to the chromatographic unit.
  • it can include a programmable microprocessor.
  • the data management system is programmed to include an algorithm and mass spectral data for identifying the signals from the detector for the individual detected compounds to convert total ion concentration values reported by the detector to what is termed an extraction ion chromatogram as a function of retention volume.
  • the programmable controller and data management systems for use in the present invention are generally those available on commercially available HPLC systems, for example, or such elements can be easily modified or reprogrammed to meet the processing requirements of the various applications of the present invention.
  • the chromatographic system of this invention can also include an eluent flow splitter in fluid communication with the eluent exit port and the detector sampling port to minimize variation in flow rate to the detector as a function of the rate of flow of mobile phase through the chromatographic unit.
  • the eluent flow splitter includes a chamber having an eluent inlet and two eluent outlets, one in fluid flow communication with a waste collection vessel through a pressure relief valve or related device and the other in fluid flow communication with the sampling port of the detector.
  • the eluent flow splitter finds particular application when the detector comprises a mass spectrometer. Such detectors often require regulated rate of flow delivered to the sample port for optimum detection efficiency.
  • the flow rate of eluent from flow splitter to the detector is a function of the setting of the pressure relief valve on the splitter and the internal diameter of the conduit or orifice communicating with the detector sampling port.
  • the chromatographic system includes a display device in communication with the data management system for reporting the detector signal as a function of retention volume.
  • the data management system can also be coupled to a data output device for reporting the k' value for at least a portion of the compounds eluted from the chromatographic unit.
  • the data management system can include a processor programmed with an algorithm for calculating other chromatographic parameters and/or descriptors (in addition to k' values) such as peak shape, peak asymmetry, median, mean, peak skewness, etc.
  • a program may be incorporated that converts point by point a chromatogram experimentally obtained under a given flow gradient profile (constant, stepped, ramped or multi-segment) into a putative chromatogram that would be obtained under other (theoretical) flow gradient conditions.
  • the chromatographic system includes at least two chromatographic units, each having an effluent flow splitter communicating with the eluent exit port and a programmable eluent switch located between the outlets (to the detector sampling ports) of each eluent flow splitter and the sampling port of the detector.
  • the eluent switch is capable of receiving eluent from each of the splitters and delivering aliquots of eluent from each of the eluent flow splitters to the sampling port of the detector, said delivered aliquots optionally separated by an aliquot of a reference fluid from a source thereof, also in fluid flow communication with the eluent switch.
  • test compounds can be screened for biological properties by combining them with a training set composition comprising one or more control compound having a common biological property to provide a test mixture.
  • a portion of the test mixture is subjected to chromatographic separation in a chromatographic system comprising a stationary phase and a mobile phase to provide numerical values characteristic of the interaction of the compounds in the test mixture with the stationary phase.
  • the numerical values of the test compounds are compared with those of the control compounds to identify the test compounds having values that best match those of the control compounds.
  • Use of the non-isorheic chromatographic system of this invention enables highly efficient data acquisition.
  • Compound purification can be accomplished using the present invention.
  • multiple test compounds are eluted from two or more chromatographic units and aliquots of the eluent are delivered to the detector via the eluent switch, which is configured so that the "waste" (i.e., that portion not delivered to the detector) is collected in a fraction collector from each respective chromatographic unit.
  • the fraction collectors are coordinated with the time of detection such that the appropriate fraction containing any detected compound can be identified.
  • the appropriate fractions containing a purified form of the identified compound are retained from the fraction collector(s).
  • the invention also finds use in the analysis, identification and purification of compounds of widely variant activity, i.e., activities ranging from pharmaceutical utility to toxicity. For example, determination of physicochemical characteristics of biologically active compounds, biologically toxic compounds and environmental toxins such as pesticides, herbicides, etc., can be accomplished using the present invention.
  • compound analysis is performed on the eluted sample compounds to determine their chemical structure.
  • compounds with predetermined physicochemical properties can be identified using the present invention.
  • the present invention provides an embodiment wherein a library of structurally related compounds is eluted from two or more chromatographic units. Aliquots of the eluent from each column are delivered to a detector capable of determining both the presence and the structural identity of the eluted compounds. The signals generated by this detector are evaluated, and elution profiles and/or other physicochemical properties of the eluted compounds are determined. In this manner, compounds with elution profiles matching the predetermined physicochemical properties desired are selected and identified.
  • the chromatographic system can include two or more chromatographic units operated in parallel using a common or independent pumps and programmable pump controllers.
  • the numerical values can be collected and stored electronically in algorithm-accessible memory for display or use in calculating related values and for performing calculations for comparing the numerical values for the individual test compounds with those in the training set or sets of compounds used to form the respective test mixtures.
  • the present invention thus provides a method for creating a database for numerical values characteristic of the interaction of solute test compounds with a mobile phase and a stationary phase in a chromatographic system, most typically in at least two chromatographic separation systems.
  • the method comprises the step of selecting a mobile phase, a stationary phase, and an initial mobile phase flow rate independently for each chromatographic separation system.
  • One or more compounds are delivered to the chromatographic separation systems for separation.
  • the flow rate of the mobile phase is changed in at least one of the chromatographic separation systems during separation of the compounds.
  • At least a subset of the compounds are detected as they elute from the column and signals indicative of compound elution, more specifically numerical values indicative of the elution volume of the eluting compound, the elution peak profile, or a value derived from said values for each detected compound is stored in a predefined data array in an electronic database.
  • the database is typically accessible by an algorithm capable of reading said data and processing it to provide information comparing the retention volume and other values characteristic of the compound's interaction with the stationary phase with those values for compounds having known biological activity.
  • the detector on the chromatographic separation system is a spectrophotometer and the eluted compounds are subjected to spectral analysis as they are eluted from the system. Numerical values derived from said spectral analysis of the eluted compounds are stored as a function of the retention volume for the eluted compounds in the electronic storage device.
  • the numeric values characteristic of the interaction of the eluted solute compounds between the mobile phase and the stationary phase in the chromatographic system typically comprises the respective retention volume of the eluted compounds or values derived from algorithmic manipulation of data relating to elution peak profiles, such values including capacity factor, peak width, standard deviations, peak skewness, peak asymmetry, peak kurtosis, and other properties that can be calculated from chromatographic peak data.
  • the method can also be conducted to produce numerical values for specific compound/stationary phase interactions, for example, by conducting chromatographic separations using mobile phases of unique pH, running multiple separations wherein the chromatographic systems are each held at unique temperatures, or running multiple separations wherein the mobile phases are of unique ionic strength. Typically the flow rate is increased during data acquisition to obtain numerical values for multiple compounds from a single injection. Fractions of the eluting compounds can be collected as part of the chromatographic separation process and thereafter the various eluting compounds can be analyzed independently for biological activity.
  • Fig. 1 is a plot of logarithm of capacity factors of a group of 13 compounds determined on an IAM column at different fractions of acetonitrile. (Barbato, F. et al., Pharm. Res.. 1997, Vol. 14, No. 12, 1677-1705).
  • Figs. 2a-b illustrate the consequences of changing compound elution order in membrane affinity fingerprinting (MAFing).
  • Fig. 3 is a diagrammatic representation of the relationship of the elements of the chromatographic system in accordance with one embodiment of the invention.
  • Fig. 4a illustrates mass chromatograms (total ion chromatogram (TIC) and Fig. 4b illustrates extracted ion chromatogram (EIC) of a mixture of drugs under isocratic flow conditions.
  • Mass chromatograms total ion chromatogram (TIC) and extracted ion chromatogram (EIC)) of a mixture of 15 drugs under isocratic flow conditions that are described in the experimental section.
  • Figs. 5a-b are similar to Figs. 4a-b showing chromatograms of the same drug mixture under gradient flow conditions.
  • Mass chromatograms total ion chromatogram (TIC) and extracted ion chromatogram (EIC) of a mixture of 15 drugs under gradient flow conditions that are described in the experimental section.
  • TIC total ion chromatogram
  • EIC extracted ion chromatogram
  • Figs. 6a-b show a comparison of capacity factors (k') from isocratic flow and gradient flow protocols.
  • Fig. 6a shows the data in tabular form
  • Fig. 6b shows a plot of the data.
  • Figs. 7a-b show a comparison of peak starting points from isocratic flow and gradient flow profiles. Gradient flow peak starting times are normalized/converted to 1 ml/min flow rate after calculation of the corresponding retention volume.
  • Fig. 7a shows the data in tabular form, while Fig.7b shows a plot of the data.
  • Figs. 8a-b show a comparison of peak ending points from isocratic flow and gradient flow profiles. Gradient flow peak ending times are normalized/converted to 1 ml/min flow rate after calculation of the corresponding retention volume.
  • Fig. 8a shows the data in tabular form, while Fig. 8b shows a plot of the data.
  • Figs. 9a-b show a comparison of peak width from isocratic flow and gradient flow profiles.
  • the base peak widths i.e., peak width at -4.4% peak height, e.g. 5 x ⁇ SD
  • the base peak width (W 44 ) is defined as the intersection of the tangents at the inflection points of the peak of interest with the baseline.
  • Fig. 9a shows the data in tabular form, while Fig. 9b shows a plot of the data.
  • Figs. lOa-b show a graphic comparison of capacity factor (k') from isocratic and gradient acetonitrile elution profiles.
  • Fig. 10a shows the data in tabular form, while Fig. 10b shows a plot of the data.
  • Figs. 1 la-b show a comparison of peak starting points from isocratic and gradient acetonitrile elution profiles.
  • Fig. 11a shows the data in tabular form, while Fig. l ib shows a plot of the data.
  • Figs. 12a-b show a comparison of peak ending points from isocratic and gradient acetonitrile elution profiles.
  • Fig. 12a shows the data in tabular form, while Fig. 12b shows a plot of the data.
  • Figs. 13a-b show a graphic comparison of peak width from isocratic and gradient acetonitrile elution profiles.
  • the base peak width (W 44 ) is the peak width determined at 4.4% peak height and is -5 x ⁇ SD .
  • Fig. 13a shows the data in tabular form, while Fig. 13b shows a plot of the data.
  • Fig. 14 illustrates the construction of a flow splitter utilized in one embodiment of the present invention.
  • Fig. 15 is a diagrammatic representation of a chromatographic system of this invention wherein 3 columns are used to generate compound data for electronic storage and later use in drug discovery protocols.
  • Figs. 16a-d comprise a graphic comparison of the capacity factors (k') of 10 compounds between 0% acetonitrile (ACN) isocratic conditions versus 5% (Fig. 16a), 10% (Fig. 16b), 20% (Fig. 16c), and 30% (Fig. 16d) ACN isocratic conditions respectively.
  • Figs. 17a-c are a graphic comparison of the capacity factors (k') of the 10 compounds used for Figs. 16a-d between flow gradient and isorheic (constant flow) conditions at 10% (Fig. 17a), 20% (Fig. 17b), and 30% (Fig. 17c) acetonitrile.
  • Figs. 18a-c are a graphic comparison of the capacity factors (k') of the 10 compounds used for Figs. 16a-d between (Fig. 18a) two isorheic runs: 2 mL/min versus 1 mL/min, (Fig. 18b) two isorheic runs: 4 mL/min. versus 1 mL/min., and (Fig. 18c) a flow gradient and an isorheic run (1 mL/min.).
  • Fig. 19 is a graphic comparison of the capacity factors (k') of the 10 compounds used for Figs. 16a-d eluted with the same mobile phase composition (10% acetonitrile) under various flow rate conditions.
  • Fig. 20 shows capacity factors for nine compounds plotted against acetonitrile concentration.
  • Fig. 21 is similar to Fig. 20 except using varying salt concentrations.
  • Fig. 22 is a plot of the capacity factors (k') of 9 compounds (obtained by flow gradient elution: ramped gradient from 0.5 mL/min. to 4.0 mL/min over 30 min.) as a function of acetonitrile fraction in the mobile phase.
  • Fig. 23 is a graphic representation of the effect of salt concentration of the mobile phase in terms of k' values for seven compounds.
  • one embodiment of the invention is based on a chromatographic system containing a pumping system, at least one injector, at least one chromatographic column, at least one pre-detector pressure relief valve
  • HPLC is the preferred chromatographic support for this invention, although other chromatographic techniques are suitable for the purpose (e.g., CEC, microchips, GC).
  • the preferred detection device is a mass spectrometer, although other detection systems, such as FTIR, FTUN and FT ⁇ MR detectors are acceptable.
  • MS/MS tandem mass spectrometer
  • the flow gradient concept was initially tested with a set of 24 compounds, which were eluted (1) under isorheic conditions (constant flow) and (2) with a gradient flow, all other chromatographic parameters being the same.
  • constant flow experiments the retention time was determined for each compound and the corresponding capacity factors were calculated.
  • each compound retention volume was determined, and the corresponding capacity factors were calculated.
  • a start point and an end point were determined and were used to calculate the peak width (W 44 ) at 4.4% peak height (i.e., 5 ⁇ ).
  • the peak start and end points were defined as the intersections of the tangents at the inflection points of a particular peak with the chromatogram baseline.
  • a chromatographic system similar to that described in Fig. 3 was used for the study.
  • the detector was either a UN spectrometer or a mass spectrometer.
  • Two HPLC Ester IAM pc c ⁇ o/c3 columns ( 30 x 4-6 mm ) were used See wo 99/10522 for a detailed description of the indicated stationary phases.
  • the mobile phase was 15% acetonitrile in 30 mM aqueous ammonium acetate (pH 7.4).
  • the sample concentration was 1 ⁇ g/ ⁇ L and the amount of sample injected was 10 to 20 ⁇ L.
  • the column pressure was up to 170 bar (or about 2400 psi) when the flow rate reached nearly 4 mL/min.
  • the choice of flow gradient profile depends on the type of analyte under consideration and its chromatographic behavior under a given set of chromatographic conditions.
  • the gradient can be ramped, stepped or multi-segment, and the gradient slopes can be steep or gentle.
  • steep gradient flows can be used for mixtures of compounds that separate well, and gentle gradient flows can be used for those mixtures with analytes that partially co-elute or have similar retention times under a given isorheic protocol.
  • the 24 test compounds were grouped essentially in two sets: compounds with low affinity for the IAM surface (17 compounds total) versus compounds having higher affinity for the surface (7 compounds).
  • Two variations of the same ramped gradient were used to elute each group of compounds.
  • the gradient was ramped from 0.5 ml/min. to 4 ml/min. over 30 min.
  • This flow gradient method was used for both UV (2 compounds: Clozapine (16) and Imipramine (17)) and MS detection (compounds (1) to (15)).
  • the second gradient flow method was identical to the first one except that after 30 min, a flow of 4 ml/min was maintained for an additional 3 hours. This flow gradient method was used for the 7 most strongly retained compounds (compounds (18) to (24), MS detection).
  • the MS system cannot tolerate high chromatographic flow because of both pumping capacity and detector sensitivity limit.
  • the advertised maximum LC flow rate tolerated by the Esquire-LC is 1.0 ml/min. Based on this consideration, when high LC flows (> 1.0 ml/min) are required for a particular study, the LC effluent needs to be split prior to entering the MS detector; thus, a piece of hardware (splitter 10), diagramed in Fig. 14 is installed immediately upstream of the MS inlet, keeping the flow to the MS detector within the range tolerated by the instrument. The excess flow is sent to a waste bottle.
  • the splitter 10 is composed of a back pressure regulator 16 (Upchurch part number P.790; 100 psi cartridge) assembled with a tee 14 (Upchurch part number P.612), and essentially functions as a pressure relief valve: the back pressure regulator 16 contains a cartridge which regulates the pressure of the incoming flow by preventing it from exceeding a given pressure (set by the manufacturer or the user). Pressure relief cartridges are available from Upchurch with the following settings: 5, 20, 40, 75, 100, 250, 500 and 1,000 psi. If more flexibility is required, the cartridges may be modified to allow the operator to manually adjust the pressure setting to a desired value.
  • the device may be applied to regulate the flow to the MS inlet in an LC/MS system operating under gradient flow conditions.
  • the Upchurch splitter 10 was connected to different ID tubings.
  • the splitter outlet 22 sending flow to the MS system is connected to a small ID tubing.
  • the excess pressure is relieved by an inline check valve 12 or pressure release valve that is linked to a larger ID tubing.
  • the pressure in the valve 12 does not exceed the preset limit, and most of the eluent flows directly from the valve inlet 20 to the outlet 22 (through a small ID tubing) in the MS system without activating the inline check valve 12.
  • the pressure is relieved by the inline check valve 12 and most of the eluent is sent via waste outlet 24 to the waste bottle through the larger tubing, and only portion of the eluent goes to the MS system via the small ID tubing.
  • the flow to the MS system was tested at two different flow rates, 0.5 and 4.0 ml/min. At 0.5 ml/min, the flow to the MS system is 0.4 ml/min. At 4.0 ml/min., the flow to the MS system is 0.8 ml/min.
  • Figs. 6a-b through 9a-b detail the test compounds used for the experiment, the chromatographic data collected under both constant and gradient flow conditions, and the associated correlation plots.
  • the concept was initially tested with Clozapine (16) and Imipramine (17) using a UV spectrometer as the detector.
  • the capacity factor (k') can be expressed as a function of retention time
  • V 0 Under isorheic (constant flow rate) conditions, V 0 can be calculated by multiplying the dead retention time (t 0 ) by the flow rate (f) (i.e., 1ml /min), and Vr is the analyte retention time (t r ) times the flow rate (f). [Note that Eq. 1 can be simplified to Eq. 2, which is commonly used by chromatographers].
  • a step gradient or any combination of the above mentioned gradient types multi-segment gradient
  • Graph (c) is to illustrate that any flow gradient profile may be used, as long as its mathematical expression is known and that the pumping system can deliver such gradient.
  • peak widths were carried out as follows.
  • the peak starting and ending points were defined as the intersection of the tangents at the inflection points of the peak of interest with the baseline of the chromatogram. This corresponds to the peak width at ⁇ 4.4% peak height.
  • These points (which define the base peak width under gradient flow conditions) were then converted (through an integration process similar to that described above) to the corresponding peak starting and ending points that would be obtained under isorheic conditions (i.e., 1.0 ml/min).
  • isorheic conditions i.e., 1.0 ml/min.
  • the base peak widths obtained with a gradient flow should be identical to those collected under isorheic conditions.
  • MathematicaTM (Wolfram Research, Champaign, IL) was used to conduct these calculations. Basically any software package that contains integration functions will be capable of calculating these data.
  • TIC Total Ion Chromatogram
  • EIC Extracted Ion Chromatogram
  • Figs. 5a-b show the corresponding data under gradient flow conditions.
  • Initial analysis of the data displayed on Figs. 4a-b and 5a-b shows that the gradient flow protocol enhances the resolution of the early eluting peaks.
  • Starting the gradient at a slower flow rate than that used for the isorheic run i.e.: ⁇ 1.0ml/min) causes the analytes with weak affinity to elute slower.
  • the method considerably reduces data acquisition time and facilitates data analysis since the peaks in chromatograms collected with a flow gradient are in general sharper and taller than those collected at lower flow rates under isorheic conditions. They are thus easier to detect, and accurate peak identification is maximized.
  • Figs. 6a-b through 9a-b unequivocally demonstrate that the chromatographic parameters obtained under gradient flow conditions are very close (virtually identical) to those collected under isorheic conditions.
  • the intercepts from these four plots are close to 0, the slopes are nearly 1 (with less than 0.040 variation), and the R-squared values are almost 1 (with less than 0.0039 variation).
  • a gradient flow chromatogram can be "converted" point by point to the corresponding constant flow chromatogram, allowing access to the chromatographic parameters (such as peak shape, peak asymmetry, skewness, kurtosis, median, mean, etc.) that are usually sought.
  • the flow gradient method was shown to enhance resolution for early eluting peaks, and to reduce data acquisition time (instrument time).
  • FIG. 15 there is provided a diagrammatic representation of a chromatographic system in accordance with this invention for determining, storing, and comparing numerical values characteristic of the interaction of one or more test compounds with a surface, a stationary phase, in the chromatographic system.
  • the illustrated system utilizes three chromatographic units, labeled columns 1, 2 and 3.
  • a pump under the control of an electronic control system delivers mobile phase past an injector point and into the chromatographic units.
  • Eluent flow splitters, S protest S 2 , and S 3 are located in fluid flow communication with each of respective columns 1, 2 and 3 and an eluent switch for delivering aliquots of eluent from each of the eluent flow splitters to the detector.
  • the splitters are also in fluid flow communication with a waste collection container through a pressure relief valve (black diamond).
  • Signals from the detector are processed in the electronic control system and directed to an output device or stored in an electronic storage database (memory), typically as a function of retention volume of the eluting/detected compound.
  • Electronic control system includes a microprocessor optionally programmed to retrieve compound data from memory and calculate related values indicative of the interaction of the eluted compounds with the stationary phase on each of the respective columns.
  • a group of 24 drugs were eluted on an este lAM.PC cl0 C3 column with a Phosphate buffer saline (PBS)-based mobile phase containing 0%, 10%, 20%, 30%, and 40% acetonitrile.
  • PBS Phosphate buffer saline
  • the retention times were plotted against the acetonitrile fraction in the PBS mobile phase.
  • Acetonitrile concentration affects the elution of the solutes in a non-linear way, and that this non-linear dependence is compound specific (different compounds are not affected to the same extent). This organic modifier effect was confirmed, as shown in Figs. 16a-d.
  • Fig. 16a The retention times at 0% acetonitrile of a group of 10 of the previous compounds were plotted against the corresponding retention times at 5% (Fig. 16a), 10% (Fig. 16b), 20% (Fig. 16c), and 30%) (Fig. 16d) acetonitrile, respectively.
  • Figs. 16a-d clearly show that if there seem to be some correlation for mobile phases that do not differ too much in composition (i.e., 0% ACN vs. 5% ACN), it fades out as the difference in acetonitrile concentration is increased (i.e., 0% ACN vs. 30% ACN). >
  • Figs. 17a-b throughl9 A group of 10 compounds were eluted under various flow rate conditions. Their respective capacity factors were determined according to the method described previously. The results of the study are represented on Figs. 17a-b throughl9. Briefly, k' values obtained under isocratic and flow gradient conditions at 10%) ACN, 20%> ACN, and 30% ACN were compared, and the correlation plots are shown on Figs. 17a, 17b, and 17c, respectively. Excellent correlation was found, with slopes and R-square values approaching 1.
  • Specific binding is the affinity exhibited between a receptor molecule and a compound wherein the receptor molecule includes a defined binding locus that discriminatorily binds those compounds which have a predetermined chemical structure. Compounds not having the predetermined chemical structure do not bind with the binding site of the receptor molecule.
  • “Compound-dependent non-specific binding” as used herein refers to that affinity interaction between a compound and a surface that does not have a specific discriminative binding locus for that compound, but rather the binding derives from the concomitant hydrophobic and/or hydrophilic interactions between the surface and the compound.
  • Non-specific binding between a surface and a compound is "compound-dependent" in that, for any one surface, different compounds will interact and bind with such surface to varying degrees based upon the chemical structure and hydrophobic/hydrophilic nature of the compound.
  • the high sensitivity of the MS detector allows the instantaneous identification of mixtures of compounds.
  • a mixture of 100 or more compounds can be injected on the column (or columns run in parallel) and detected as they elute from the chromatographic system. In theory, depending on the loading capacity of the columns, a mixture of up to 1000 compounds can be analyzed.
  • the data from the MS analysis will be correlated to that of the UV detectors connected to the chromatographic system, resulting in the assignment of a retention time and capacity factor for each and every compound detected.
  • the data can be collected electronically and used as input for the calculation of one or more physicochemical values according to predetermined algorithms or equations.
  • the present invention can be applied to the rapid and efficient collection of databases of physicochemical values and/or biologically relevant parameters for large compound libraries. Consequently it seems perfectly suited for lead identification and optimization of chemical libraries, which is a very important aspect of the drug discovery process, as well as QSAR studies. Once a "hit" compound has been identified, derivatization by the usual combinatorial chemistry tools to a large number of structurally similar parent molecules is possible.
  • the present invention provides a convenient and efficient technique for the analysis of this pool of derivatives and the identification of one or more compounds with a data set of physicochemical values (derived from the chromatographic system) that would classify the compounds of interest as potential promising new leads.
  • An obvious example of application of the technique is separation conditions optimization. Since a correlation between the retention volume of a given compound and the gradient flow profile used to elute it can be established, an algorithm similar to that proposed by Jinno et al. can be developed to predict the elution behavior of said compound under various gradient conditions. An initial chromatographic run of a compound mixture under a chosen flow rate profile, coupled with concomitant identification of the eluent, would establish the retention volume for each component in the mixture. This experimental data can then be used to simulate the elution profile of said compound mixture under various flow gradient conditions, allowing the user to establish the expected flow gradient conditions necessary for optimal resolution and separation of the compounds.
  • the present invention may be particularly well suited for the study and optimization of separation conditions for any one mixture of compounds.
  • Other examples of applications are drug analysis/screening: evaluation of compounds put in contact with a surface suitable for pharmacokinetic and pharmacodynamic studies.
  • the invention may also find applications in the field of diagnostics: physiological fluids sampling (such as blood or urine) for specific compounds that may be diagnostic of some disease or condition, or for metabolic studies, may be performed.
  • physiological fluids sampling such as blood or urine
  • the present invention may also be relevant to environmental sampling (water, soil analyses for contamination) and quality control in the food industry for example (e.g., flavors, ingredients, preservatives, etc.).

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Abstract

La présente invention concerne un procédé et un appareil qui permettent d'établir une base de données comprenant une caractéristique de valeurs de données de l'interaction de composés et de surfaces, telles que des phases stationnaires d'un système chromatographique. Selon cette invention, on utilise un gradient de flux de phase mobile pour renforcer l'efficacité et la précision des données dans des protocoles de criblage de médicament à haut rendement. Ce système chromatographique comprend un séparateur (10) de flux éluant en communication fluidique avec l'unité chromatographique, et le détecteur destiné à minimiser la variation du débit du flux vers le détecteur avec la variation du débit du flux de la phase mobile est situé dans cette unité chromatographique. Par l'utilisation d'un détecteur capable de fournir un signal de présence, ou de préférence une caractéristique d'identification, de composés d'élution en provenance de ce système, cette invention offre un procédé puissant permettant de créer une base de données de caractéristique de valeurs numériques de l'interaction de composés test de soluté avec une phase mobile et une phase stationnaire dans au moins deux systèmes de séparation chromatographique. On peut générer efficacement des données et les stocker dans des systèmes de stockage électronique accessibles par algorithme (bases de données) en vue de les utiliser seules ou en association avec d'autres descripteurs moléculaires destinés à la prédiction de l'activité biologique.
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WO2009062538A1 (fr) * 2007-11-12 2009-05-22 Agilent Technologies, Inc. Système de hplc à débit variable
DE102007063440A1 (de) 2007-12-21 2009-06-25 Thomas Grimm Screeningsystem zur Durchführung und direkten Analyse von biologischen, biochemischen und chemischen Synthese- und Umsetzungsreaktionen
EP2421631A4 (fr) * 2009-04-23 2013-12-04 Xcellerex Inc Système et procédé pour charger un système de chromatographie à commande par rétroaction à vitesse variable
US20180284079A1 (en) * 2017-03-30 2018-10-04 Shimadzu Corporation Liquid chromatograph
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EP1552302A4 (fr) * 2002-07-24 2006-09-13 Ciphergen Biosystems Inc Cartographie de la difference d'interaction entre des proteines
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WO2009062538A1 (fr) * 2007-11-12 2009-05-22 Agilent Technologies, Inc. Système de hplc à débit variable
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US9618485B2 (en) 2007-11-12 2017-04-11 Agilent Technology, Inc. HPLC-system with variable flow rate
DE102007063440A1 (de) 2007-12-21 2009-06-25 Thomas Grimm Screeningsystem zur Durchführung und direkten Analyse von biologischen, biochemischen und chemischen Synthese- und Umsetzungsreaktionen
EP2421631A4 (fr) * 2009-04-23 2013-12-04 Xcellerex Inc Système et procédé pour charger un système de chromatographie à commande par rétroaction à vitesse variable
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