Liquid purification or separation – With means to add treating material – Chromatography
Reexamination Certificate
2000-11-13
2002-07-23
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
With means to add treating material
Chromatography
C210S659000, C210S198200
Reexamination Certificate
active
06423220
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a dry chromatography method for the purification of from 1 mg to a few hundred mg quantities of compounds, to the type of columns and to the nature of the sorbents to be used for such separations. The method is suitable for purification of several compounds simultaneously, e.g. the purification of chemical libraries. As described in the present invention the method is well suited for automation since addition of the sample can be done in a simple and reproducible manner and since the eluent is simply added to the top of the column and in the exact amount needed for the separation.
BACKGROUND OF THE INVENTION
Combinatorial chemistry and high speed synthesis has become very important tools in medicinal chemistry. Large “libraries” of compounds are synthesised and subjected to High Throughput Screening (HTS) trying to identify novel structures with interesting biological activity. A “chemical library” may contain several thousands of compounds.
The chemical libraries may be prepared by conventional solution chemistry but solid phase methods are also being used. Many different types of automated synthesisers have been developed and automation is indeed a very important factor when trying to handle large numbers of compounds simultaneously. The compounds are commonly prepared in amounts of 1-100 mg, most often about 5-20 mg.
The quality of the library is highly dependent on the purity of the individual compounds present in the library. When a set of chemical reactions are applied to different substrates the yields of reaction may differ significantly, the end result being a chemical library where the purity of the compounds may vary considerably. Obviously, a simple method for the purification of several compounds at the same time would be very attractive. The method should preferably be suited for automation.
HPLC systems equipped with automated injectors can handle the separation of several samples. However the samples are separated sequentially on the same column and the column has to be regenerated after each run. Large amounts of solvents are needed and contamination of the column may often be a serious problem preventing long time use. Also, an extreme number of different fractions need to be collected and handled.
Dry column chromatography is a relatively old chromatographic method (Reviews: B. Loev and M. M. Goodman. in “Progress in Separation and Purification” 1970; vol. 3: 73-95. F. M. Rabel in M. Zeif and R. Speights (Eds.), “Ultrapuification”, Dekker, New York 1972, p 157). The mixture to be purified is added to the top of a dry column of e.g. silica and the column is developed with a suitable solvent. When the solvent reaches the end of the column the separated compounds are located (e.g. by colour), the column cut into pieces and the desired compounds isolated by extraction. Typically column sizes of diam. 1-3 inches and length 10-20 inches have been used for purification of compound mixtures of several hundred mg to several grams. The particle size used has commonly been >63&mgr;.
Dry column chromatography has not been utilised for the simultaneous purification of large numbers of compounds, e.g. the purification of chemical libraries, and has never been subjected to automation in this connection.
Dry column chromatography has not been recognised as a suitable method for rapid purification of mg quantities of compounds on short columns.
As mentioned above dry column chromatography is an old chromatographic method and today it is practically never used. Obviously the advantages that are obtained when it is performed as described in the present invention have not been realised.
SUMMARY OF THE INVENTION
The object of the present invention is to eliminate the shortcomings and solve the problems stated above in connection with the known techniques for simultaneous purification of several compounds in a sample. This object is achieved by a chromatografic column of the type described by way of introduction having the features specified in the characterising part of claim 1. These and other features and advantages will appear from the following description and the subclaims. The present invention also relates to a method for simultaneous purification of several compounds in a chromatography column according to the present invention and also to use of such a column.
DESCRIPTION OF THE INVENTION
The present invention relates to a dry chromatography column to be used for rapid purification of from about 1 mg to a few hundred mg quantities of compounds. Several separations can be run simultaneously and the columns are very well suited for use in automated procedures.
According to the. present invention it has been found that short dry chromatography columns can be prepared by packing columns of suitable plastic materials with sorbents of small particle size, and that these columns can be used for rapid purification of small amounts of material. The top of the column contains an empty volume. Methods are provided whereby addition of the sample can be done in a simple and reproducible manner. The eluent is thereafter added to the top of the column and in the amount needed for the separation. The simplicity and the reproducibility of the chromatographic method described in the present invention make it well suited for automation. When the separations have been completed the desired compounds can be located on the columns, that particular part of the column cut out, and the compounds isolated by extraction.
Compared to conventional HPLC methods dry column chromatography utilises only small amounts of solvents and does not require handling of very large numbers of different fractions.
The columns of the present invention can be made of different plastic materials. These materials should be transparent or semitransparent to UV and visible light, to allow for location of the separated compounds on the column. The plastic should be compatible with the solvents to be used for the separations. Since the compounds are isolated by cutting out the desired part of the column it is important that the plastic is easy to cut with minimal deformation of the sorbent. Suitable thicknesses are between 0.5 to 3 mm, preferably 1 to 2 mm. Since the columns of the present invention are short they are still rigid enough to be easy to handle. Cutting can be done with a sharp object such as a knife, razorblade and the like. The columns are preferably cylindrical but other forms may be used.
Suitable plastic materials are those which can be used together with different organic solvents e.g. polyethylene, polypropylene and Teflon. Teflon has superior chemical resistance and is soft to cut. Polyethylene is softer than polypropylene. If very soft plastic materials are used, such columns may be put inside an outer shell. Combinations of plastic materials may also be used.
For the separations to proceed with acceptable speed the length of the column is generally less than 20 cm, preferably less than 15 cm. Columns of small diameter are easier to cut without deformation than wider columns. The diameter of the column may generally vary from about 0.3 to about 3 cm, preferably less than 1.5 cm. However, smaller columns may be used if almost analytical separations are desired and larger columns may be of use for separations of large amounts of material. For large amounts of material it may be more advantageous to use several columns in parallel. The problem that a compound moves slightly faster near the column wall is of greater significance for wider columns (c.f. B. Loev and M. M. Goodman. in “Progress in Separation and Purification” 1970; vol. 3: 73-95).
The column should have an empty volume above the sorbent preferably large enough to be able to hold the volume of solvent needed to develop the column. For a silica column this volume is generally 0.5 to 0.8 times the volume holding the sorbent. The volume may be indicated by a mark, a helpful feature when adding the developing solvent.
It is very important that the sample to be purified can be added to
Smith , Gambrell & Russell, LLP
Therkorn Ernest G.
Trikonex AB
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