Liquid purification or separation – Processes – Chromatography
Reexamination Certificate
2000-08-25
2002-05-14
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
Processes
Chromatography
C210S659000, C210S143000, C210S198200, C436S161000
Reexamination Certificate
active
06387273
ABSTRACT:
FIELD OF THE INVENTION
The present invention generally relates to the purification and/or characterization of libraries of compounds, for example, combinatorial and/or lead generation libraries.
BACKGROUND OF THE INVENTION
Currently, there are many general methods of purifying synthetic compounds. These methods generally involve purifying a single target compound from multiple impurities.
Compounds are currently being prepared in relatively large numbers in combinatorial and lead generation libraries. Often, compounds are synthesized in multi-well plates or multi-tube arrays, with the number of related compounds numbering in the thousands. The compounds are then typically purified using HPLC.
Combinatorial libraries are synthesized using a variety of solvents and solvent mixtures. It is difficult to perform high performance liquid chromatography (HPLC) on samples prepared in multi-tube arrays without first removing the solvents, because the solvents often cause the compounds to elute in the void volume of the column. Additionally, use of the solvents can cause compounds to precipitate on the column head or injector or transfer lines, which can lead to plugging the inlet, increasing the column back pressure, and restricting the flow through the column. Further, to the extent that it may be desirable to use a single gradient for purifying compounds on an HPLC column, it is difficult, if not impossible, to define a standard protocol when the solvents used to prepare the compounds can vary from compound to compound.
Because of these limitations, the solvents used for the synthesis are typically removed prior to purifying the compounds, and the compounds are then re-dissolved in a second solvent system prior to adding the compound to the HPLC column. Choice of the proper solvent for dissolving the sample is a special problem in preparative HPLC, because the goal is to inject a relatively large amount of sample into a preparative HPLC column without having the sample precipitate in the column. While this method works, it adds two time consuming steps—removing a first solvent, and adding a second solvent.
It would be advantageous to have methods for purifying compounds on an HPLC column without having to remove the solvent prior to placing the compounds on the HPLC column. The present invention provides such methods.
SUMMARY OF THE INVENTION
Methods for purifying and/or characterizing compounds, particularly libraries of compounds such as combinatorial or lead generation libraries, are disclosed. Purification devices capable of being used in the method are also disclosed.
Compounds are commonly synthesized via combinatorial chemistry, typically in multi-tube racks. The amount of solvent in each of the test tubes in the racks is typically less than about 2 mL. The methods described herein permit one to purify compounds directly from the vessel in which they are prepared without first removing the solvent used to prepare the compound.
The methods involve equilibrating the HPLC column with a first mobile phase such that, when the sample is added to the column in the solvent in which it is prepared, the sample does not significantly elute off of the column in the void volume, and the solvent in which the compound is prepared does elute off of the column in the void volume or shortly thereafter. Then, after the solvent has eluted, an appropriate purification protocol can be used to purify the compound, for example, using gradient elution.
There are several parameters which can be varied to arrive at a mobile phase such that the compound of interest does not significantly elute in the void volume or cause blockage in the system. First, the solvent power can be adjusted such that the mobile phase is not a strong solvent for the compound of interest. For example, when a reverse phase column is used, the type of compounds which most rapidly elute off the column are polar organic compounds. When the compounds are organic compounds, water is a reasonable solvent for use in the mobile phase. However, the water preferably includes a minor amount (preferably between 1 and 10 percent v/v, more preferably between 2 and 6 percent, most preferably between 3 and 5 percent) of a water-miscible organic solvent.
A second way to avoid having the compound of interest elute in the void volume or cause blockage in the system is to decrease the size of the void volume, having first ensured that the mobile phase in the void volume is not a strong solvent for the compound of interest. The void volume can be decreased, for example, by using a relatively short column and asorbent with a particle size of about 5 microns or less, with suitable inlet and outlet tubing. Typically, HPLC is conducted using devices in which the tubing has an internal diameter of about 0.01 inches. The tubing used in the methods described herein preferably has an internal diameter between 0.01 and 0.04, and more preferably, between about 0.02 and 0.03 inches. This allows the column to have significantly less back pressure and less chance of blockage caused by precipitation of compounds in the column inlet and outlet while using a different sample solvent than the mobile phase.
A third way to avoid having the compound of interest elute in the void volume or cause blockage in the system is to saturate the column with a weak solvent prior to sample injection, for example, by increasing the flow rate of a mobile phase which is a weak solvent for the compound of interest through the HPLC column. Next, one can apply a suitable solvent gradient system until the solvent used to prepare the compound of interest has eluted off the column, while retaining the compounds of interest on the column.
A fourth way to avoid having the compound of interest elute in the void volume or cause blockage in the system is to inject samples in a relatively large volume of lower concentration rather than as a relatively small volume of a higher concentration.
A fifth way to avoid having the compound of interest elute in the void volume or cause blockage in the system is to decrease the particle size of the column packing (sorbent) while using a relatively short and relatively fat column with suitable inlet and outlet tubing sizes to handle the increased pressures.
Preparative HPLC is commonly performed using a sorbent particle size of about 10 &mgr;m or larger with a long column. Preferably, the methods herein use a sorbent with a particle size of about 5 microns or less, more preferably with a relatively short column (for example, 50 mm or less).
The methods herein can use one or more of the above, in any suitable combination, to minimize elution of the compound of interest in the void volume or precipitation of the compound in the HPLC system. Preferably, the solvent used to prepare the compound is a water-miscible organic solvent. If it is not, use of a co-solvent is preferred. Co-solvents are organic solvents which are water-miscible, and can cause the non-water-miscible solvent to become miscible in the water. The amount of sample volume which can be applied to a typical preparative HPLC column is an important but imperfectly understood operation. Normally samples should be injected in the form of a relatively large volume at a lower concentration rather than in the form of a smaller volume at a higher concentration. The amount of sample volumes which can be applied to a typical preparative HPLC column is about 2 mL or less.
In order to determine an appropriate set of conditions for purification a combinatorial library, one can perform route scouting on the library to determine which set of conditions described above is appropriate for allowing the solvent used to prepare the compound(s) to elute off the column in the void volume or shortly thereafter while the compound(s) of interest does not significantly elute off the column in the void volume or cause blockage in the system.
DETAILED DESCRIPTION
Methods for purifying and/or characterizing compounds, particularly libraries of compounds such as combinatorial or lead generation libraries, are disclosed. Purif
Burns Doane Swecker & Mathis L.L.P.
Scynexis Chemistry & Automation, Inc.
Therkorn Ernest G.
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