JP2002502031A - A rapid method for separating small molecules using reversed-phase high-performance liquid chromatography - Google Patents

Abstract

The present invention is a rapid method for separating small organic compounds using gradient reverse phase HPLC. The method of the present invention achieves a resolution with a run time of less than 1 minute and peak production of at least 1 peak / 2 seconds. The method can also achieve separation of a compound from a mixture of compounds in an elution sample having a volume of 2 milliliters or less.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the field of high performance liquid chromatography, and more particularly, to the field of high performance liquid chromatography separation of small organic molecules.

BACKGROUND OF THE INVENTION High performance liquid chromatography (HPLC) is commonly used for analytical and preparative separations of biopolymers and other organic molecules. For example, individual components within a complex organic reaction mixture can be separated by HPLC. HPLC is performed in a pressure tube containing the fixed adsorbent that is the packing material. A pressure mechanism applies pressure to the mobile phase applied to one end of the column, causing it to move through the column and exit to the opposite end of the column. A sample containing the mixture of compounds is injected onto the column through the sample injection port. As the sample moves through the packing material, the various components of the sample will have different affinities,
Adsorb to this packing material. Thus, these components can be separately eluted from the column under appropriate conditions. In a reverse phase HPLC column, compounds in a sample are separated based on hydrophobicity.

[0003] HPLC analysis can be performed in isocratic or gradient mode. Fixed composition HP
LC separation is performed under a constant eluent composition. Gradient HPLC separation
Due to the gradual change in the proportion of two or more solvents applied to this column over time,
Characterized. This solvent change is often controlled by a mixing device that mixes solvent A and solvent B, producing the HPLC solvent just before it travels to the column. The amount of time that the slope changes from one extreme to the other extreme is the slope time.

[0004] Generally, in gradient chromatography, increasing the flow rate and / or decreasing the gradient time increases the resolution (ie, the ability of the column to separate components within the mixture into separate eluent fractions). ) Is believed to cause losses.

[0005] Rapid methods for creating combinatorial libraries and preparing and isolating potential drug candidates using automated synthetic organic chemistry techniques represent a significant advance in drug discovery. Certain combinatorial libraries include a series of compounds that have common structural features but differ in the number or type of groups attached to their primary structure. Each compound in the combinatorial library formed by parallel synthesis is a separate sample contained in a tube or well of a microtiter plate. Once the library is complete, each sample undergoes quality control analysis to confirm that the particular sample contains the desired library components at the required purity. Generally, this is accomplished by subjecting these samples to HPLC with UV, evaporative light scattering detection (ESLD), or mass spectrometry detection; IR; NMR; or any other suitable analytical technique. . Qualitative analysis of such combinatorial libraries by conventional HPLC requires an average of 5 to separate the various compounds in this sample.
Need ~ 20 minutes.

[0006] A problem encountered with conventional methods of separating compounds in combinatorial libraries using HPLC is the length of time required to separate each sample. Each sample of a combinatorial library created by parallel synthesis must be analyzed separately to determine whether the sample contains the appropriate compound and / or to separate the compounds in this mixture. Must. Each library contains a large number of samples, each of which requires an average of 10 minutes of run time. The amount of time required to perform a separation on these samples can be monthly, using standard equipment and methodology.

SUMMARY OF THE INVENTION [0007] The present invention provides a method for analyzing a relatively simple synthetic mixture containing a small number of reagents, desired product (s) and relatively few by-products using HPLC. A rapid method of isolation for preparative separation is provided. The method of the present invention can be used without significant loss of resolution.
The average run time of the HPLC analysis per sample is 5 to 20 times that found in the prior art.
From 1 minute to less than 1 minute. The present invention, in part, minimizes the total volume of eluent applied to the column, maximizes the linear flow rate of the eluent, and compresses the gradient time to separate peaks at least every 2 seconds. By doing so, it relies on the discovery that small organic molecules can be separated by full gradient reverse phase HPLC. Total gradient is defined as a change in solvent B concentration of at least 50%. For example, if the initial concentration of solvent B was 15%, when the concentration of solvent B reached 65%, a full gradient would be achieved. If the total eluent volume is reduced and the flow rate is increased to the level indicated in the present invention, the resolution of the peak eluted from this column is such that a mixture of small organic compounds cannot be clearly distinguished and separated. Until then, it was believed in the prior art that it would be significantly reduced.

[0008] The method of the invention involves applying the mixture of compounds to a reversed phase column designed with a gradient high performance liquid chromatography system and operating at a flow rate of at least 5 column volumes / minute. This column contains 1
A complete gradient is applied at a rate using a maximum of 0 column volumes, preferably 5 column volumes. These parameters allow each small organic component in the mixture of compounds to be eluted from the column in a different fraction with sufficient resolution to allow at least 1 peak / 2 seconds of peak production. Become. A 1 minute analysis with 1 peak / 2 second peak generation is interpreted as an analysis that can baseline separate more than 30 individual peaks.

[0009] The amount of time required for a complete separation depends on the parameters used in this separation (eg, the length of the column and the amount of solvent used). Preferably, the mixture of compounds is applied to the column at a first time point, and all compounds are eluted within less than 1 minute from the first time point. In another preferred embodiment, all compounds are eluted in less than 30 seconds. In another embodiment, all compounds are 2
It is eluted in less than 0 seconds.

In one embodiment of the invention, the method also includes detecting at least one of these compounds as it elutes from the column. In another embodiment, the method comprises collecting at least one of the compounds in different fractions as it elutes from the column.

In a preferred embodiment, the mixture of molecules contains a reactant and a substantially pure product of the reactant.

In other preferred embodiments (including those listed above), the column comprises 30
mm or less. This column is, in another embodiment, no longer than 15 mm.

According to other preferred embodiments (including those listed above), the column comprises:
It has a filler material with an average diameter of less than 5 microns.

In other preferred embodiments (including those listed above), the peak generation is
At least 1 peak / 1 second. This peak generation is, in another embodiment, at least 1 peak / 0.5 second.

Preferably, the total volume of liquid applied to this column per analysis is less than 15 column volumes, preferably less than 8 column volumes. The total volume of the liquid may contain a wash volume with up to twice the column volume. In a preferred embodiment, the total volume of the liquid may contain an equilibrium volume with up to one column volume.

In other embodiments (including those listed above), the mixture of molecules is a large combinatorial library of small organic molecules. Preferably, the combinatorial library is created by a parallel synthesis method, and the method is performed for high-throughput purification and / or quality control analysis.

In a further embodiment, the flow rate of the column is a linear velocity of at least 3 mm / sec. In another embodiment, the linear velocity is 5 mm / sec.

The small organic compounds eluted from the column are typically analyzed by a detection device such as a UV detector. In one embodiment of the present invention, the small organic molecule is UV
It is analyzed by both the detector and the mass spectrometer.

In one embodiment, the method is a method for analyzing at least one compound in a mixture of the compounds. Preferably, less than 10 μg of this mixture of compounds is applied to the column for this analysis. In one embodiment, a sample of the compound is not collected for further use after eluting it from the column.

In another embodiment, the method is for preparative isolation of at least one compound in a mixture of the compounds. Preferably, between 1 mg and 100 mg of this mixture of compounds is applied to the column for this analysis. In one embodiment,
These compounds are collected in separate fractions after they are eluted from this column for further use. Preferably, the at least one compound is collected in a fraction having a volume of no more than 2 milliliters.

[0021] The high performance liquid chromatography can, in one embodiment, be performed at a temperature greater than 20 ° C. In another embodiment, the method is performed at a temperature greater than 50C. In a preferred embodiment, the method is performed at a temperature greater than 60C.

In another aspect, the present invention is a rapid high performance liquid chromatography method for isolating a concentrated fraction of small organic compounds from a mixture of compounds for preparative separation. The method comprises the steps of: applying a mixture of compounds to a reversed phase column in a gradient high performance liquid chromatography system, wherein the column has a flow rate of at least 5 column volumes / minute; Applying a full gradient to this column at a maximum volume of 10 column volumes to elute these small organic compounds in different fractions, this elution allowing resolution with a peak generation of at least 1 peak / 4 seconds To separate from other molecules from the column, and to collect the small organic compound. The small organic compound is, in one embodiment, collected in a fraction having a maximum volume of 2 milliliters.

In other embodiments (including those listed above), the mixture of molecules is a large combinatorial library of small organic molecules. Preferably, the combinatorial library is created by a parallel synthesis method, which is performed for high-throughput purification and / or quality control analysis.

Each of the limitations of the present invention can encompass various embodiments of the present invention. Thus, it is expected that each of the limitations of the invention, including any one element or combination of elements, may be included in each method.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel method for separating small organic molecules using reverse phase HPLC, with applications for analysis and / or preparative isolation of separated compounds. I do. Although the principles underlying analytical and preparative HPLC are the same (ie, separating the mixture into distinct components), traditionally, the modes of operation are different. The goal of analytical HPLC is focused on using the minimum amount of material for optimal resolution, whereas preparative chromatography is loaded with the largest amount of material that can be separated satisfactorily. You are focused on doing. Further, in preparative chromatography, the eluate is collected after separation, since the isolation of certain components is important. The dual nature of the procedure was reasonable when the analytical or preparative isolation was for a small number of compounds, in which case each analysis could be customized. However, using this same method to analyze and / or purify a very large number of different compounds synthesized by parallel synthesis is too costly and time consuming. In order to maintain high throughput operation for both analytical and preparative isolation, a faster new method of the present invention has been developed, wherein the differences between the analytical and preparative methods are: , Just the size of the equipment. By appropriately increasing the column size and flow rate, the preparative column can be used to achieve 1-10 μg loading, as can be achieved with an analytical column.
By loading 0 mg, the same separation with the same resolution can be achieved.

As used herein, an “analytical method” for a small organic compound is a method in which a sample of a small organic compound is loaded onto a column and performed according to the methods of the invention described herein. It is. In this method of analysis, the sample may or may not be collected. In a preferred embodiment, when performing the method of analysis, the sample is not collected. Preferably, the analytical method comprises loading the column with less than 20 μg,
More preferably, the method includes the step of loading a sample of less than 10 μg.

As used herein, a “preparative isolation method” for a small organic compound refers to a method in which a sample of a small organic compound is loaded onto a column according to the method of the invention described herein; The method performed, wherein the compounds are collected in fractions as they elute from the column. Preferably, the preparative isolation method comprises the step of loading the column with less than 100 mg, more preferably between 1 mg and 100 mg of sample.

The novel method of the present invention encompasses both analytical and preparative separations and is significantly faster than conventional methods. The method of the present invention is particularly advantageous for separating components of a low molecular weight combinatorial library as part of quality control analysis and / or purification often performed for such libraries. Prior to the present invention, each sample of a particular combinatorial library was approximately 5-20 minutes for separation by HPLC.
Minutes needed. Using the method of the invention, it has surprisingly been found that the separation time per sample can be reduced to less than one minute. This reduction in time significantly increases the number of samples that can be separated per unit time per instrument.
At a minimum, the present invention reduces the separation time by a factor of five compared to conventional methods, allowing separation of at least five times more compounds. In a preferred embodiment, these methods are more than 10 times faster than conventional methods. Using conventional methods, which typically require a 10 minute total HPLC run time in a fully automated facility, approximately 2,000 samples per month can be separated. Using the method of the present invention, which requires only a one minute run time, 20,000 samples can be separated in the same time. This new method is described in detail below.

FIG. 1 illustrates instruments useful according to the general method of the present invention. The two solvent reservoirs 12 and 14 containing solvents A and B are pumps 10 and 10
A allows the mixing chamber 16 to pass through the tubes 18a and 18b, respectively.
Pumped up to. Computer 32 controls the amount of solvent A and B pumped into this mixing chamber over time. Solvents A and B are mixed in this mixing chamber to form a homogenous solvent, which passes from tube 18c through high pressure valve 19 to column 20 (which contains a reversed phase packing material called the stationary phase). ). A plurality of samples are contained in a microtiter plate 24 mounted on an automatic injector 22 (also connected to this switching valve by a pipe 18f). The sample in each well of the plate 24 is conveyed through the switching valve via the pipe 18f, and is then conveyed to the column by the automation means through the pipe 18d. Once the sample enters the column, the small organic compounds in the sample adsorb to the stationary phase with different affinities based on the hydrophobicity of the compound. After the sample is loaded, the ratio of solvent A to solvent B is shifted over time to form a solvent gradient through the column. At some point in the gradient, different small organic molecules are eluted from the column and carried to the detector. This eluent passes through a detection device, such as a UV detector 28,
The compounds in this eluate are characterized. In some cases, the eluent is exposed to multiple detectors (eg, UV detector and mass spectrometer 30). If isolation of a particular component is important, the eluent from the UV detector will consist of a small portion (which goes to the mass spectrometer via tube 18g) and a major portion (which 18
via i to the fraction collector (31)). The equipment required to carry out the invention can be assembled from commercially available equipment. Although some of these devices have in fact been designed for other functions related to HPLC analysis, they can easily be adapted to the functions described herein. For example, in a preferred embodiment, the 50 μl mixing chamber shown for mixing the eluent in the assay setting is in fact a chamber commonly used for post-column derivatization purposes. In addition, other equipment assembled to manufacture this equipment is used in accordance with a preferred embodiment of the present invention, and is described in further detail below to manufacture equipment useful for performing the methods of the present invention. Adjusted to

[0030] The method of the present invention is based, in part, on the small organic molecules that minimize the volume of liquid applied to the column, maximize the linear flow rate, and reduce the gradient time. By compressing to produce a peak generation of at least 1 peak / 2 seconds,
It can be analyzed and purified by full gradient reverse phase HPLC. Prior to the present invention, manipulating these parameters above the levels described in the prior art would reduce the resolving power of the peaks eluted from this column to obtain individual isolates of a mixture of small organic compounds. It was believed that to a degree that would not be possible, it would drop significantly. For example, combinatorial libraries containing small organic molecules often require about 5 to 2 for quality control analysis.
Separated by gradient reverse phase HPLC using a run time of 0 minutes. According to the prior art, reducing this total run time by reducing the column volume and increasing the linear flow rate was believed to result in an equivalent loss in resolution. Surprisingly, according to the present invention, this run time is reduced by the minimum described in the prior art, based on the peak volume, only minimizing the loss of resolution, if the gradient time is also shortened. It has been discovered that the execution time can be reduced by a factor of five. This finding means that the method of the present invention can produce faster separations than those found in the prior art, with minimal loss of peak volume. The peak volume parameter is defined as the number of baseline separated peaks that fit within the time that the gradient changes from a low percentage of solvent B to a high percentage of solvent B. To normalize between different analytes,
The peak volume divided by the slope time is referred to as the "peak product".
ion) "and expressed in units of peak / second. Using the method of the present invention, separation can be at least 1 peak / 2 seconds. Using the method of the present invention,
Preferably, separation is performed at 1 peak / 1 second, more preferably at 1 peak / 0.5 second. Resolving power is the ability to identify individual compounds eluted from this column. Sufficient resolution according to the present invention is the ability to resolve one peak every 2 seconds. As used herein, this means that the peak width at baseline is less than 2 seconds on average. Determination of resolution using measurement of baseline peak width is described in L.W. R. Snyder, J .; J. Kirkland, J .; L. G
Lajch, "Practical HPLC Method Development.
ment, 2nd Edition, John Wiley & Sons, Inc. (1997
), The entire contents of which are incorporated herein by reference.

As is apparent from FIG. 1 and the above description, several variables affect the peak generation performance of the method. Among these are (1) the size of this column,
(2) packing material used in this column, (3) use of full gradient with minimum volume, (
4) the total volume of liquid passing through the column, and (5) its linear flow rate. These variables should be adjusted as described further below to produce a fast method with a maximum run time of 1 minute and peak generation of at least 1 peak / 2 seconds.

In a preferred embodiment, the columns used according to the method of the invention are short and wide. Preferably, the column has a length of less than 30mm. Shorter columns allow for higher flow rates. With longer columns, the flow rates must be reduced to minimize the back pressure created in the column. In addition, wide columns (e.g., columns having an inner diameter greater than 4 mm) are preferred to minimize extra column band widening associated with other components of the instrumentation. However, according to the method of the present invention, columns having any width can be used. Preferred widths for analytical HPLC are between 4 mm and 5 mm. The preferred width for preparative isolation is between 20 and 30 mm.

The packing material used in this column is solid support particles having reversed phase properties. Preferably, the filler material has a particle size of less than 5 μm, more preferably
It has a particle size of less than 4 μm. Such packing materials are commercially available and are well known to those skilled in the art. It is possible to develop improved packing materials, in which case the preferred particle size can vary depending on the improvement of the material.

[0034] The appropriate volume of solvent applied to the column is an important parameter for the method of the present invention. Typically, for a full gradient HPLC analysis, about 30 column volumes of solvent are applied to the column, and often higher amounts are applied for preparative isolation. The maximum volume used in accordance with the present invention is 15 column volumes, preferably 8 column volumes, to maximize speed. A graph depicting the maximum volume of liquid applied in accordance with the method of the present invention, in units of column volume, is presented in FIG. Volumes used in conventional manner are presented in parentheses below the volumes of the present invention. As shown in this figure, a full gradient is applied to this column within 10 column volumes with a time lapse of 20-40 seconds. Conventional gradients require 15 to achieve full gradients.
~ 30 volumes are required. A two column volume wash cycle of 95% acetonitrile or other elution solvent is used to flush any residual molecules from the column over a period of about 5-10 seconds. The column is then equilibrated within one column volume and subjected to initial solvent conditions for one column volume for a total time of about 5-15 seconds.

The preferred mixing volume of this solvent is the minimum volume that allows for a homogeneous mixture of solvent A and solvent B. This minimum volume is achieved by using a small volume mixing chamber and minimum volume piping. A mixing chamber suitable for this analytical embodiment has an initial volume of less than 250 μl, preferably less than 50 μl. Although such small mixing chambers are not commercially available, 50 μl post-column reaction chambers are commercially available and can be used as mixing chambers. In addition to selecting the mixing chamber with the smallest size, it is preferable to fix this mixing chamber. As used herein, a stationary mixing chamber is a chamber or column packed with beads, which are usually made from steel or glass. As the solvent migrates into the beads, it undergoes turbulence and mixes together.

It is important that the method of the present invention be performed using a complete gradient to ensure that all components injected on this column are removed from the column before the next sample injection. It is. As used herein, "complete gradient"
"dient)" is a gradient of solvent starting with a low proportion of solvent B (solvent B is a non-polar solvent such as acetonitrile) and a high proportion of solvent A (solvent A is its aqueous phase). is there. The low proportion of solvent B is preferably lower than 20%. The proportions of solvents A and B are shifted over time, producing a high proportion of solvent B and a low proportion of solvent A. The high proportion of solvent B is preferably greater than 70%.

[0037] The flow rate and the mixing volume dictate the time these two solvents reach the HPLC column (much less than 0.1 minutes in our analytical system). 50
Using a μl mixing chamber, minimum tubing, and flow rate of 3-5 ml / min, the transfer of the solvent from the pump to the column is completed in 2-6 seconds. Another important parameter is the linear flow rate (ie, the rate at which the solvent moves through the column). The linear flow rate depends on the flow rate and the inner diameter of the column.
Preferably, this linear velocity is higher than 3 mm / sec.

An additional parameter that can be used to increase peak volume is temperature. As demonstrated in the examples below, when the temperature of the sample through the continuous liquid flow path is higher than 20 ° C., the peak volume increases significantly. When combined with the high flow rates of the present invention, performing this method at elevated temperatures provides a significant increase in peak volume. Thus, while the method of the invention may be performed at any temperature suitable for HPLC analysis, higher temperatures may be preferred to increase the peak volume of the system. In one embodiment, the temperature is 20C, 30C, 40C, 50C.
Or it may be at a temperature of 60 ° C. or higher. When choosing an appropriate temperature for the HPLC method, one skilled in the art will realize that some compounds may be unstable at elevated temperatures. Those skilled in the art
Compounds that are unstable at high temperatures can be identified.

The method of the present invention is useful for separating small organic compound mixtures. This mixture of compounds may contain a large number of small organic compounds of unknown composition with differences in hydrophobicity. The method of the present invention is capable of separating at least 30 compounds exhibiting different hydrophobic properties in less than 1 minute, as confirmed by the gradient composition in which each compound elutes. Under conditions where the separation power of this method results in a peak production of 1 peak / 1 second, the method of the present invention is capable of separating about 60 such samples in one minute.

Usually, the mixture of compounds contains a much smaller number than the maximum number of compounds that can be separated by the system. For example, a preferred mixture of compounds contains the reactants and the substantially pure products of these reactants. As used herein, a mixture of compounds containing "substantially pure products of these reactants" refers primarily to the intended product, small amounts of unreacted starting material as well as a small number (preferably 5 A mixture containing by-products (less than species) (in significantly lower amounts than the intended product). Such a mixture is achieved by using reactants that, under given reaction conditions, can only yield a limited number of products. In a preferred embodiment of the invention, the mixture of these compounds is derived from the preparation of a combinatorial library. A "combinatorial library of small organic compounds" is a collection of closely related analogs, which differ from one another in one or more points of diversity, and are synthesized by organic techniques using multi-step processes. It is. Combinatorial libraries contain a vast number of small organic compounds, some of which may have significant biological activity.

One of the preferred combinatorial libraries implemented according to the present invention is prepared by means of a parallel synthesis method resulting in a compound array. As used herein, "
A "compound array" is a collection of compounds that are identifiable by spatial address in Cartesian coordinates and are arranged such that each compound has a common nucleus and one or more variable structural diversity elements. The compounds in such a compound array are produced in parallel in separate reaction vessels, and each compound is identified and tracked by its spatial address. Regardless of the relative amounts of starting materials and products present in each reaction vessel after the reaction, the method of the present invention analyzes the extent of the reaction and / or
Alternatively, they can be used to isolate these components. Examples of parallel synthesis mixtures and methods are described in U.S. patent application Ser. S. S. N. 0
8 / 177,497 and its corresponding PCT published patent application WO 95/1897.
2 (which was published on July 13, 1995) and US Pat.
No. 2,171 (registered Jan. 27, 1998) and its corresponding PCT published patent application WO 96/22529, the contents of which are incorporated herein by reference. ing.

Once a series of small organic compound mixtures have been developed, these mixtures can be separated and analyzed to determine the products formed and the extent of the reaction. Each mixture of compounds represents a separate sample injected on the HPLC column. As shown in FIG. 1, each sample is held in a well of a microtiter plate 24 in an automatic sampling device 22. These samples are loaded and injected manually or automatically using equipment controlled by computer 32. Automatic loading and injection of these samples is preferred as it allows for continuous loading of samples at a rate that does not limit the overall analytical process.

Before loading the sample onto the column, the column is conditioned by flowing the intended mobile phase through it. As noted above, the column is packed with a non-polar stationary phase on solid support particles and can be obtained from various commercial sources.
HPLC columns are available from various commercial sources (eg, MACMOD (Chaddsfor)
d, PA)). Once the column is conditioned, the system is started by injecting the sample. As the small organic compounds come into contact with the non-polar packing material, each molecule is adsorbed to the packing material. The affinity with which each compound adsorbs this packing material depends on the hydrophobicity of the individual compound. The injection of this sample is defined as time zero for this operation.

Immediately after injection of the sample, a gradient is applied to the column to elute the bound compounds in different fractions in the column. Gradient HPLC system
Contain reservoirs 12 and 14, each of which includes a mixing chamber 16 by a pump.
Containing different polar solvents pumped through onto column 20. In a preferred embodiment, the solvent stream is a non-pulsating HPLC pump (eg, Shimadz).
u (Colombia, MD) available as part of the HPLC system)
Is maintained by The column is then applied with a total gradient of 15% to 95% acetonitrile. Of the compounds in the mixture injected into the column, those compounds that are polar have a greater affinity for the initial composition of the eluent than the stationary phase, and therefore, It elutes more rapidly than high compounds, which have a greater affinity for this hydrophobic stationary phase. Reverse phase HPL
Solvents typically used for the gradient of C include acetonitrile, methanol,
Isopropanol and propanol. Modifiers are typically added to the mobile phase, primarily to buffer its pH to a narrow range, including various acids and bases, such as phosphoric acid. , Perfluorinated carboxylic acids and amines).

When small organic compounds are eluted from the column, an HPLC compatible detector is used to detect their presence. A compatible detector is one that can detect a signal derived from a compound in the eluent and generates a signal indicating the presence of the compound. This detector has 1
At a speed of more than 10 points per second, preferably more than 20 points per second,
Enable data acquisition. HPLC compatible detectors include fluorescence, electrochemical, IR,
Examples include, but are not limited to, NMR, chemiluminescence, UV and mass spectrometry. Preferably, the HPLC compatible detector is a UV detector 28. This is because commercial UV detectors can achieve the required data acquisition speed when their settings are adjusted to obtain the maximum value. In addition, UV detectors are generally applicable to many different chemical classes and diverse mobile phases.

In some cases, the HPLC compatible detector is both a UV detector 28 and a mass spectrometer 30. The use of both a UV detector and a mass spectrometer is preferred.
This is because it allows the method of the present invention to obtain both purity (by UV) and structural (by MS) information for each separated and detected component eluted from this column. In addition, the mass spectrometer can be used as a highly specific multi-dimensional detector to provide general information about certain compounds or specific information about particular compounds. These beneficial properties, coupled with the inherently high sensitivity of mass spectrometers, make it one of the most desirable detectors to combine with the resolving power of HPLC.

In one aspect of the invention described above, the method may be used for analysis (eg, quality control analysis) and / or preparative isolation of at least one component of a mixture of compounds. In this aspect of the invention, after separation on the column, one or more compounds may be collected in different fractions. This HPLC compatible detector is used to confirm the presence of compound in each fraction, and these fractions are collected in an acceptable container (eg, tube,
Or wells of a microtiter plate).

In another aspect, the present invention is a rapid high performance liquid chromatography method for preparative isolation of a concentrated fraction of small organic compounds from a mixture of compounds. Each of the parameters described above in connection with this separation method is also applicable to this aspect of the invention.
The method according to this aspect of the invention, however, is characterized in that the separation of this molecule is carried out solely for the purpose of separating the compounds in the mixture into different fractions, and that these compounds can be further analyzed or used. Is different from the above method in that
The method is performed as described above, except that the eluent allows for a resolving power with a peak production of at least 1 peak / 4 second. In some embodiments,
This eluent allows for resolution with a peak production of at least 1 peak / 2 seconds.

[0049] Preferably, these compounds are collected in fractions having a volume of 2 mL or less. The method of the present invention achieves compound separation in a short time such that the total volume of each compound eluted is 2 mL or less. This is advantageous because, in contrast to the prior art methods, the target compound is present in a considerably concentrated form, in which case the target compound is often eluted in a minimum volume of 4 mL.

As noted above, many variations on these particular examples are possible and, therefore, these examples are merely illustrative of the invention and are not limiting.

EXAMPLES Example 1 Gradient Reversed-Phase HPLC Analysis of a Standard Mixture of Small Organic Molecules Apparatus: All analytical separations were run on Shimadzu (Columbia, MD) H
PLC System (this consists of two LC-10AS pumps, SIL-10A
Automatic sampling device, SCL-10A system controller and SPD-10AU
V-detector). This system was modified for high speed applications in the following manner. A low volume static mixer from Supelco (Bellefonte, PA) equipped with a 250 μl cartridge was used to mix the eluate quickly and efficiently under high pressure. In combination with a short 30 mm column, a 50 μl mixing cartridge was used to further minimize gradient delay. The initial bypass of this Shimadzu auto-injector is to eliminate band broadening effects.
Removed. Injections were performed in a 50 μl loop, injecting between 1 μl and 5 μl in partially filled loop mode. Connections between the injector, column and detector were made using 0.007 "tubing and their length was kept to a minimum to prevent extra column band broadening. The initial flow cell of the instrument was replaced with a semi-micro version, which has a path length of 5 mm and a smaller total volume of 2.5 μl, the response of this detector being such that a narrow peak with rapid elution could be detected. Thus, it was set to 1. Data acquisition was performed at a speed of 20 points / second by Ju.
Chr manufactured by sice Innovations (Palo Alto, CA)
This was performed with the software impact.

Chemicals: Water and acetonitrile are HPLC grade, dimethylsulfoxide was obtained from Baker (Philippsburg, NJ). C. S. It was grade. All other chemicals are available from Aldrich (Milwaukee, W.).
It was the highest purity grade available from I). These HPLC eluates are
. Prepared by adding 1% (v / v) trifluoroacetic acid to water (solvent A) and acetonitrile (solvent B).

HPLC column: Zorbax SB-C8 column (50 mm × 4.6 mm and 30 mm × 4.6 mm, both packed with 3.5 μm particles);
150 mm × 4.6 mm (filled with 5 μm particles)) is a MACMOD
(Chaddsford, PA). Prontosil C18-SH
The column (50 mm × 4.6 mm, 3 μm particles) was a Bischoff Anal.
The gift was from ysentechnik (Leonberg, Germany).

One example of a large difference in compound hydrophobicity is represented by our standard test mixture, which consists of the following components: acetamidophenol, 2-hydroxydibenzofuran and t-butylphenoxybenzaldehyde. For reverse phase HPLC separation, the acetamidophenol elutes during the early stages of the gradient and the 2
-Hydroxydibenzofuran elutes at an intermediate stage of the gradient and the t-
Butylphenoxybenzaldehyde elutes at the end of this gradient. These three
A mixture of the various compounds was prepared in dimethyl sulfoxide (DMSO). This mixture was then subjected to HPLC analysis under the conditions described above according to the method of the present invention.

The test mixture was first added to each of the above conditions at a flow rate of 3 ml / min.
. Analysis was performed using a column packed with reversed phase silica having a particle size of 5 μm.
The compounds were eluted with a gradient of 15-95% acetonitrile, which was applied to the column in 0.7 minutes, with a retention time of 10 seconds and an equilibration time of 5 seconds. The result is shown in FIG. Three major peaks, which represent each of the three components of the test mixture, separated in less than one minute. Based on the baseline peak width of the middle peak, about 60 compounds having different hydrophobicities were
In this way the baseline could be separated.

The same mixture of compounds was added to each of the above conditions at a flow rate of 5 ml / min.
Analysis was performed using a column packed with reversed phase silica having a particle size of 5 μm. These molecules were again subjected to a gradient of 15-95% acetonitrile, which was applied to the column in 0.3 minutes, with a retention time of 10 seconds and an equilibration time of 5 seconds.
Eluted. The result is shown in FIG. Again, based on the width of the middle peak, about 30 compounds with different hydrophobicities could be baseline separated in this 30 second method.

Example 2 Gradient Reversed-Phase HPLC Analysis of Combinatorial Library Samples Obtained by Parallel Synthesis Two different combinatorial libraries (this was a U.S. application filed Jan. 5, 1994)
. S. S. N. 08 / 177,497 and its corresponding PCT published patent application W
O95 / 18972, which was published on July 13, 1995, and US Pat. No. 5,712,171, which was registered on January 27, 1998.
(Prepared by parallel synthesis as in the method disclosed in WO 96/22529 and its corresponding PCT published patent application) was selected for analysis using the method described in Example 1. The results for sample A1990702D9 analyzed using a traditional 20 minute analysis are compared in FIG. 5 with those obtained from the 1 minute analysis and the 30 second analysis. The 20 minute analysis was performed at 15 ml / min with Zorbax SB-C8 (150 x 4.6 mm) packed with 5 μm particles and a complete gradient (1 x 1 mm).
5% to 95% of solvent B) was used. Although the peak volume is smaller for the shorter analysis, the resolution is sufficient to determine purity even for such complex samples (this is the worst case scenario). Is). Similarly, sample AQ130QC48H5 using 20 minute analysis, 1 minute analysis and 30 second analysis
Are shown in FIG. Once again, this one minute method provides the same purity information as the longer 20 minute analysis. In addition, this 30 second analysis provides similar information. The small peak between the two major peaks is partially merged with the major component, but still has sufficient resolution to indicate the presence of the minor component. It is believed that the details set forth above are sufficient to enable one skilled in the art to practice the invention. The disclosed embodiments are intended only as exemplifications of particular embodiments of the invention enabled herein, and that any method that is functionally equivalent is within the scope of the invention. The invention is not limited in scope by these embodiments. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Example 3 Effect of Flow Rate on Rapid Gradient Reversed-Phase HPLC Analysis of the Present Invention To determine the effect of flow rate on the rapid analytical method of the present invention,
Performed using various flow rates ranging from ４4 ml / min. The data is shown in FIG. In the case of a very steep one-minute gradient, increasing the flow rate from 2 ml / min (producing a gradient volume of 2.4 column volumes) to 4 ml / min (4.8 column volumes) will reduce the peak volume by 29 To 48. The even greater gradient volume achieved by increasing this flow rate also shortens its total analysis time, as indicated by the elution of the last peak. The peak generation rate in this case almost doubles at a flow rate of 0.47-0.8 peaks / second. The linear flow rate at 4 ml / min is 6.4 mm / sec,
The back pressure was about 250 bar, which is acceptable for normal use. Therefore, the column can be operated at very high linear flow rates with steep gradients to obtain high peak volumes and peak production rates.

Example 4 Gradient Reversed-Phase Preparative HPLC Analysis of a Standard Mixture of Small Organic Molecules Apparatus: Preparative separation consists of two Rainin Dynamax SD-1 pumps (Woburn, Mass.), Gilson-215 (Madison, WI), which is configured as both an automatic sampler and a fraction collector, Shi.
Madzu (Colombia, MD) SPD-10A UV detector (this is
(With a preparative cell). Injections were performed in partially filled loop mode, which is a 5 ml loop and injects 250 μl. Detection was at 254 nm, and the detector response was set to 1 to allow detection of narrow peaks eluting quickly. Data acquisition is based on Gilson (M
(adison, Wis.).

Chemicals: Water and acetonitrile are HPLC grade and dimethylsulfoxide was obtained from Baker (Philippsburg, NJ). C. S. It was grade. All other chemicals are available from Aldrich (Milwaukee, WI).
) Was the highest purity grade available. These HPLC eluates were used at 0.
1% (v / v) trifluoroacetic acid was added to water (solvent A) and acetonitrile (solvent B).
) Was prepared.

HPLC column: YMC ODS-A C18, 20 m packed with 5 μm particles
m × 50 mm and obtained from YMC (Wilmington, NC).

A standard test mixture of acetamidophenol, 2-hydroxydibenzofuran and t-butylphenoxybenzaldehyde was added at 40 mg / m in DMSO.
1 for each component. This mixture was then subjected to preparative HPLC analysis. The mobile phase flow rate was 80 ml / min and the compounds were eluted with a gradient of 10% to 95% acetonitrile applied to the column for 1 minute (20 sec retention time and 40 sec equilibration time). . The fraction collector was set to collect its second and third peaks. FIG. 8 shows the result. Based on its baseline peak width, approximately 30 compounds with different hydrophobicities could be separated and collected using this method.

Example 5: HPLC Analysis for Gradient Reversed-Phase Preparing of Combination Library Samples Combination library (USSN filed January 5, 1994)
. 08 / 177,497 and its corresponding PCT published patent application WO 95/18.
972, which was published on July 13, 1995, and U.S. Pat.
712,171 (registered January 27, 1998) and prepared by parallel synthesis by methods such as those disclosed in the corresponding PCT published patent application WO 96/22529. One sample was selected for analysis using the method described in Example 4. However, the detection was performed at 217 nm and 400 μl of a 30 mM solution of this sample was injected. The fraction collector was set to collect the expected product peak, and this peak was identified as the last peak in the chromatogram. The result of sample A1990703D9 is shown in FIG.

Example 6: Effect of Temperature on Rapid Gradient Reversed-Phase HPLC Analysis To determine the effect of temperature on the rapid analysis method of the present invention, the method was performed, for example, under conditions of different flow rates. Using various temperatures ranging from 20 ° C to 60 ° C,
Carried out. The data is presented in Table I. As shown in this table, the peak volume increases significantly when the temperature is increased to 20-60 ° C., and the increase in peak volume is even more pronounced when operating the system at high flow rates. . As shown in this table, 30 m at 4 ml / min using a 1 min method with a 0.75 min gradient.
The peak volume of the sample run on the m column rose from 44 at 25 ° C to 55 at 60 ° C. This correlates with a 25% increase based on a corresponding decrease in peak width. In a similar experiment in which the flow rate and temperature were increased from 4 ml / min to 5 ml / min and 25 ° C. to 60 ° C., respectively, the peak volume increased from 44 to 60, a 35% increase. In addition to this change in peak volume, the eluate viscosity decreases at higher temperatures. Faster flow rates with similar pressure drops can also be achieved at higher temperatures. For example, when the temperature was increased from 20 ° C. to 60 ° C., a 50% pressure drop was observed. In addition, a 5-10% increase in eluate rate was observed, indicated by a decrease in retention time of the compound.

[0065]

[Table 1]

During experiments related to this temperature effect, it was discovered that without some improvements, existing equipment could not effectively perform the appropriate temperature changes. First, a column heater jacket was used to control the temperature. This column is immersed in a temperature-controlled circulating liquid. The eluate was passed through a heat exchange capillary, which was immersed in a thermostated system, to the desired temperature. However, the temperature of the eluate was found to drop from about 50 ° C. to about 28 ° C. between the mixer and the column at the high flow rates required for the rapid HPLC analysis of the present invention. . A modification of this apparatus was arranged by immersing the manual injector valve, along with the heat exchanger and column, in a water bath at the desired temperature for the experimental conditions. This improvement to the device provided a uniform temperature distribution throughout the liquid path and allowed the characterization of the temperature effects. One of ordinary skill in the art, based on this teaching, can modify existing equipment to achieve these effects. All references, patents, and patent publications discussed in this application are incorporated by reference throughout this specification.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of instruments for performing the method of the present invention.

FIG. 2 shows the HPL by the column volume of the eluent applied to the column.
3 is a graph depicting the composition of the mobile phase during C analysis.

FIG. 3 is a chromatogram of a test mixture obtained using the method of the invention with a total analysis time of less than 1 minute.

FIG. 4 is a chromatogram of a test mixture obtained using the method of the invention with a total analysis time of less than １ ／ minute.

FIG. 5 is a comparison of chromatograms obtained for samples synthesized by parallel synthesis using 20 minute, 1 minute and 30 second analysis.

FIG. 6 is another example of a comparison of chromatograms obtained for samples synthesized by parallel synthesis using analyzes at 20 minutes, 1 minute, and 30 seconds.

FIG. 7 shows a 50 × 4.6 m, 3 μm Prontosil C18-SH column: (7A) 2 ml / min flow rate; (7B) 3 ml / min flow rate; and (7C) 4 m
5 is a series of chromatograms obtained using the method of the present invention to determine peak volumes obtained at various flow rates using a flow rate of 1 / min.

FIG. 8 is a chromatogram from a preparative isolation of 10 mg of each of three standard compounds using a 20 mm × 50 mm column packed with 5 μm particles and a flow rate of 80 ml / min.

FIG. 9 is a chromatogram from a preparative isolation of approximately 5 mg of a mixture prepared by parallel synthesis using the conditions identified in FIG.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission Date] January 27, 2000 (2000.1.27)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0004

[Correction method] Change

[Correction contents]

[0004] Generally, in gradient chromatography, increasing the flow rate and / or decreasing the gradient time increases the resolution (ie, the ability of the column to separate components within the mixture into separate eluent fractions). ) Is believed to cause losses. (Sny
der, L .; Et al., "Practical HPLC Method Level."
operation ", Wiley-Interscience Publicati
on, (1997)).

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0007

[Correction method] Change

[Correction contents]

SUMMARY OF THE INVENTION [0007] The present invention provides a method for analyzing a relatively simple synthetic mixture containing a small number of reagents, desired product (s) and relatively few by-products using HPLC. A rapid method of isolation for preparative separation is provided. The method of the present invention can be used without significant loss of resolution.
The HPLC analysis execution time per sample was determined by the conventional technique (Weller et al., Mole).
Culcular Diversity, (1997), 3: 61-70) from an average of 5-20 minutes to less than 1 minute. The present invention provides, in part,
By minimizing the total volume of eluent applied to the column, maximizing the linear flow rate of the eluent, and compressing the gradient time to separate peaks at least every 2 seconds, small organic molecules are Full gradient reverse phase HPLC
Relies on the discovery that they can be separated by Total gradient is defined as a change in solvent B concentration of at least 50%. For example, if the initial concentration of solvent B is 15%
If the concentration of this solvent B reaches 65%, a full gradient is achieved.
If the total eluent volume is reduced and the flow rate is increased to the level indicated in the present invention, the resolution of the peak eluted from this column is such that a mixture of small organic compounds cannot be clearly distinguished and separated. Until then, it was believed in the prior art that it would be significantly reduced.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0028

[Correction method] Change

[Correction contents]

The novel method of the present invention encompasses both analytical and preparative separations and is significantly faster than conventional methods. The method of the present invention is particularly advantageous for separating components of a low molecular weight combinatorial library as part of quality control analysis and / or purification often performed for such libraries. Prior to the present invention, each sample of a particular combinatorial library was approximately 5-20 minutes for separation by HPLC.
(Weller et al., Molecular Diversity).
, (1997), 3: 61-70). Using the method of the invention, it has surprisingly been found that the separation time per sample can be reduced to less than one minute.
This reduction in time significantly increases the number of samples that can be separated per unit time per instrument. At a minimum, the present invention reduces the separation time by a factor of five compared to conventional methods, allowing separation of at least five times more compounds. In a preferred embodiment, these methods are more than 10 times faster than conventional methods. Using conventional methods, which typically require a 10 minute total HPLC run time in a fully automated facility, approximately 2,000 samples per month can be separated. Using the method of the present invention, which requires only a one minute run time, 20,000 samples can be separated in the same time. This new method is described in detail below.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0030

[Correction method] Change

[Correction contents]

[0030] The method of the present invention is based, in part, on the small organic molecules that minimize the volume of liquid applied to the column, maximize the linear flow rate, and reduce the gradient time. By compressing to produce a peak generation of at least 1 peak / 2 seconds,
It can be analyzed and purified by full gradient reverse phase HPLC. Prior to the present invention, manipulating these parameters above the levels described in the prior art would reduce the resolving power of the peaks eluted from this column to obtain individual isolates of a mixture of small organic compounds. It was believed that to a degree that would not be possible, it would drop significantly. For example, combinatorial libraries containing small organic molecules often require about 5 to 2 for quality control analysis.
Separated by gradient reverse phase HPLC using a run time of 0 minutes. According to the prior art, reducing this total run time by reducing the column volume and increasing the linear flow rate was believed to result in an equivalent loss in resolution. Surprisingly, according to the present invention, this run time is reduced by the minimum described in the prior art, based on the peak volume, only minimizing the loss of resolution, if the gradient time is also shortened. It has been discovered that the execution time can be reduced by a factor of five. This finding means that the method of the present invention can produce faster separations than those found in the prior art, with minimal loss of peak volume. The peak volume parameter is defined as the number of baseline separated peaks that fit within the time that the gradient changes from a low percentage of solvent B to a high percentage of solvent B. To normalize between different analytes,
The peak volume divided by the slope time is referred to as the "peak product".
ion) "and expressed in units of peak / second. Using the method of the present invention, separation can be at least 1 peak / 2 seconds. Using the method of the present invention,
Preferably, separation is performed at 1 peak / 1 second, more preferably at 1 peak / 0.5 second. Resolving power is the ability to identify individual compounds eluted from this column. Sufficient resolution according to the present invention is the ability to resolve one peak every 2 seconds. As used herein, this means that the peak width at baseline is less than 2 seconds on average. Determination of resolution using measurement of baseline peak width is described in L.W. R. Snyder, J .; J. Kirkland, J .; L. G
Lajch, "Practical HPLC Method Development.
ment, 2nd Edition, John Wiley & Sons, Inc. (1997
).

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0041

[Correction method] Change

[Correction contents]

One of the preferred combinatorial libraries implemented according to the present invention is prepared by means of a parallel synthesis method resulting in a compound array. As used herein, "
A "compound array" is a collection of compounds that are identifiable by spatial address in Cartesian coordinates and are arranged such that each compound has a common nucleus and one or more variable structural diversity elements. The compounds in such a compound array are produced in parallel in separate reaction vessels, and each compound is identified and tracked by its spatial address. Regardless of the relative amounts of starting materials and products present in each reaction vessel after the reaction, the method of the present invention analyzes the extent of the reaction and / or
Alternatively, they can be used to isolate these components. Examples of parallel synthesis mixtures and methods are described in U.S. patent application Ser. S. S. N. 0
8 / 177,497 and its corresponding PCT published patent application WO 95/1897.
2 (which was published on July 13, 1995) and US Pat.
No. 2,171 (registered Jan. 27, 1998) and its corresponding PCT published patent application WO 96/22529.

[Procedure amendment 6]

[Document name to be amended] Statement

[Correction target item name] 0065

[Correction method] Change

[Correction contents]

[0065]

[Table 1]

[Procedure amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0066

[Correction method] Change

[Correction contents]

During experiments related to this temperature effect, it was discovered that without some improvements, existing equipment could not effectively perform the appropriate temperature changes. First, a column heater jacket was used to control the temperature. This column is immersed in a temperature-controlled circulating liquid. The eluate was passed through a heat exchange capillary, which was immersed in a thermostated system, to the desired temperature. However, the temperature of the eluate was found to drop from about 50 ° C. to about 28 ° C. between the mixer and the column at the high flow rates required for the rapid HPLC analysis of the present invention. . A modification of this apparatus was arranged by immersing the manual injector valve, along with the heat exchanger and column, in a water bath at the desired temperature for the experimental conditions. This improvement to the device provided a uniform temperature distribution throughout the liquid path and allowed the characterization of the temperature effects. One of ordinary skill in the art, based on this teaching, can modify existing equipment to achieve these effects.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), AU, CA, JP, US (72) Inventor Kiranos, James N.M. United States Massachusetts 02170, Wollaston, Philips Street 99

Claims (34)

[Claims] 1. Applying a mixture of compounds to a reversed phase column (20) in a gradient high performance liquid chromatography system; applying a full gradient to said column (20) at maximum volume; An improved rapid method for separating each small organic compound in a mixture of said compounds by eluting small organic compounds from said column (20) into different fractions, said improvement comprising at least 5 column volumes / min. Using a flow rate and eluting each small organic compound such that the elution allows resolution with peak production of at least 1 peak / 2 seconds, wherein the maximum volume is: The method wherein the column volume is 10 times. 2. The method according to claim 1, wherein at least one of said compounds is provided on said column (20).
2. The method of claim 1, further comprising the step of detecting as it elutes from. 3. The method according to claim 1, wherein at least one of said compounds is provided on said column (20).
2. The method of claim 1, further comprising the step of collecting in different fractions as they elute from. 4. The method of claim 1, wherein the mixture of molecules comprises a reactant and a substantially pure product of the reactant. 5. The method according to claim 1, wherein the column (20) has a length of 30 mm or less. 6. The method of claim 1, wherein said column (20) has a packing material having an average diameter of less than 5 microns. 7. The method of claim 1, wherein said peak generation is at least 1 peak / 1 second. 8. The method according to claim 1, wherein the peak generation is at least 1 peak / 0.5 second.
The method of claim 1. 9. The total volume of liquid applied to the column is a 15-fold column (2
0) The method of claim 1, wherein the volume is less than 0). 10. The method of claim 1, further comprising a wash volume having up to twice the column volume. 11. The method of claim 1, further comprising an equilibrium volume having up to one column volume. 12. The method according to claim 1, wherein the mixture of compounds is added to the column (20) at a first time point.
The method of claim 1, wherein all the compounds are eluted within less than 1 minute from the first time point. 13. The method of claim 12, wherein all compounds are eluted in less than 30 seconds. 14. The method of claim 12, wherein all compounds are eluted in less than 20 seconds. 15. The method of claim 1, wherein the mixture of molecules is a multi-combination library of small organic molecules. 16. The method according to claim 15, wherein the combination library is created by a parallel synthesis method. 17. The method of claim 1, wherein said column has a linear velocity of at least 3 mm / sec. 18. The method of claim 1, wherein said method is a method for analyzing at least one compound in a mixture of said compounds. 19. The method of claim 2, wherein said method is a method of analyzing at least one compound in a mixture of said compounds. 20. The method of claim 1, wherein said method is a preparative isolation method for at least one compound in a mixture of said compounds. 21. The method of claim 3, wherein said method is a preparative isolation method for at least one compound in a mixture of said compounds. 22. The method of claim 3, wherein said at least one compound is collected in a fraction having a volume of 2 milliliters or less. 23. A mixture of compounds is applied to a reversed phase column (20) in a gradient high performance liquid chromatography system; a full gradient is applied to said column (20) at maximum volume; And from the column (20),
An improved rapid high performance liquid chromatography method for preparative isolation of a concentrated fraction of said small organic compound from a mixture of said compounds by separating said small organic compound from other molecules; and collecting said small organic compound; The improvement includes using a flow rate of at least 5 column volumes / min and eluting the small organic compound such that the elution allows for a resolving power having a peak production of at least 1 peak / 4 seconds. The method wherein the maximum volume is 10 column volumes. 24. The method of claim 23, wherein the mixture of molecules comprises a reactant and a substantially pure product of the reactant. 25. The method according to claim 23, wherein the column (20) is 20-30 mm long. 26. The method of claim 23, wherein the column (20) has a packing material having an average diameter of less than 5 microns. 27. The method of claim 23, wherein the total volume of liquid applied to the column (20) is less than 15 column volumes. 28. The method of claim 23, further comprising a wash volume having up to twice the column volume. 29. The method of claim 23, further comprising an equilibrium volume having up to one column volume. 30. The method of claim 23, wherein all compounds are eluted in less than 60 seconds. 31. The method of claim 23, wherein said mixture of molecules is a multi-combination library of small organic molecules. 32. The method according to claim 31, wherein the combination library is created by a parallel synthesis method. 33. The method according to claim 23, wherein said column (20) has a linear velocity of at least 3 mm / sec. 34. The method of claim 23, wherein said small organic compound is collected in a fraction having a maximum volume of 2 milliliters.