Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
  • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
  • B01D15/1864—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns
  • B01D15/1885—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns placed in parallel
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
  • B01D15/16—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the fluid carrier
  • B01D15/163—Pressure or speed conditioning
  • B01D15/165—Flash chromatography
• • 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
  • G01N30/26—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
  • G01N2030/326—Control of physical parameters of the fluid carrier of pressure or speed pumps

Definitions

• the present invention relates to a process for flash chromatographic separation for purification of chemical compounds, which is very suitable for handling several separations simultaneously.
• the invention also relates to equipment for such separations, including a chromatography device . Background of the invention
• High Throughput Screening (HTS) of large compound collections has become an important tool for the pharmaceutical and biotech industry when attempting to identify new potential drug molecules. Analysis of the biological screening data is naturally dependant upon the quality of the compounds being tested and the advantage of having compounds of high purity is increasingly being realised.
• the compound collections often include compound libraries prepared by parallel synthesis. When preparing many compounds simultaneously there is of course a need for simple, rapid, reliable and economical purification methods including chromatography.
• the object of the present invention is to provide a simple and highly economical chromatographic process for purification of chemical compounds. Another object is to provide equipment for such separations.
• Fig. 1 shows the underlying principle of the chroma- tography method according to the present invention.
• Fig. 2 shows how several separations are performed simultaneously according to the present invention.
• the present invention involves a novel process for preparative flash chromatographic separations highly suitable for rapid parallel purification which operates without the need for solvent pumps and pressure.
• the invention is accomplished by connecting one end of a tubing to the bottom, i.e. the lower end, of a chromatographic column. When the tubing becomes filled with solvent this creates an effect of suction driving the eluent through the column. According to the invention it was found that the chromatographic performance using this technique was similar to that when applying pressure on top of the column.
• the flow rate can be adjusted (see Fig.
• a typical separation is performed as follows: Having a column positioned as in Fig. 1A, a portion of eluting solvent (eluent) is added to the column. Typically about half the column is thus “wetted” .
• the mixture to be separated is dissolved in a small amount of eluent and added to the column, after which a small amount of eluent may be used to "wash down" the sample before filling the column with eluent .
• the upper loop of the tub- ing is positioned above the top of the column, i.e.
• Said upper loop represents a part of the tubing having the ability to be positioned at or above the eluent surface in the column. More than one part of said tubing may be positioned in such a way, e.g. in a wave form.
• the chromatography may be initiated by amending the position of the chromatography column and/or said part of the tubing in such a way that said part is positioned below the eluent surface of the column.
• the tubing outlet during chromatography always has to be positioned below the lower end of the column. This means that several columns can be prepared in advance and that the chromatography can be started simultaneously by raising the column (or lowering the tubing outlet) as in Fig. IB.
• the above-mentioned initial "wetting" step with eluent is optional.
• the columns are packed with chromatographic materials of small particle size in order to achieve good separations. Typically the mean particle size is ⁇ 60 ⁇ . Improved separation properties were observed for silicas with a mean particle size ⁇ 40 ⁇ .
• the chromatographic material is positioned between filters of a suitable material, e.g. polyethylene, teflon etc. Reservoirs, preferably of a transparent material e.g polypropylene, with a filter at the bottom are filled with the desired amount of chromatographic material and finally another filter is placed on top.
• the height of chromatographic material in the columns should be quite short in order to obtain useful flow rates, typically below 10 cm and preferably below 5 cm, especially for chromatographic materials of small particle size. Since wider columns give higher flow rates than more narrow ones, the column width influences which packing heights are useful .
• the amount of packing material naturally depends on the application, the difficulty of the separation and the amount of sample to be separated.
• the chromatographic material is silica 0.5 g - 5 g would be used to purify 2 - 200 g of sample and 5 g - 50 g for 0.2 - 2 g. In case of simpler separations higher loadings of sample may be used.
• chromatographic materials may be used for the chromatographic method described in the present invention, including silica, modified silicas, alumina etc. Low cost materials which can be used for disposable columns are especially useful .
• a fluorescent material may be mixed into the chromatographic material making it possible to visualise the separation by observing the column in UV-light. Thus, fractions can be collected when the desired compound (s) leave the column. It is also possible to see when all compounds have left the column, thus stopping the elution and minimising the solvent use.
• Fluorescence Indicator green 254 nm can be mixed into the silica in various amounts, typically 0.5 - 5%.
• the empty space above the packing material should be so large that it can hold all, or most of, the solvent needed for the separation, typically 2 to 10 times the dead volume of the column, especially 3 to 6 times.
• the length of the tubing is such that the distance h can be varied to obtain desired flow rates.
• the distance h may be between 10 and 150 cm, or more typically between 10 and 75 cm, when the chromatographic procedure is performed in a conventional fume hood.
• the inner diameter of the tubing may influence the flow rate such that thin tubings reduces it .
• the tubing may be connected to the column outlet or via some inter- mediary holder, such as a holder which can accommodate several columns (c.f . Fig 2) .
• the tubing may also be connected via a valve .
• Special equipment and devices can be designed to perform chromatography according to the present invention and is within the scope of the invention.
• Such equipment is characterised by the following properties.
• the vertical distance between the columns and the tubing outlets can be adjusted in order to obtain desired flow rates.
• the end or outlet of the tubing, or the above-mentioned part(s) thereof, can be positioned above the surface of the eluting solvent (c.f. Fig 2) such that all separations can be started simultaneously.
• the latter feature may be replaced by the use of individual valves connected to each column, especially when only few separations are started at the same time.
• the number of separations to be performed simultaneously can vary widely and is for practical reasons dependant upon the size of the columns, the number and size of the fractions to be collected, and the nature of the fraction collector. In cases of rapid separations the fraction collection is easily done by hand without the need for an automated fraction collector.
• 96 well plates for fraction collection can be very useful in which case optimally 8 or 12 separations are performed simultaneously.
• a polypropylene reservoir (25 ml, inner diameter 20 mm) was equipped with a polyethylene filter and packed with 3.0 g of silica (6-35 ⁇ from Grace Davison and mixed with 4% fluorescence indicator) and another filter placed on top and pressed against the chromatographic material .
• the height of silica was 19 mm.
• Ethyl acetate was added to the column, without connection to tubing, and the flow rate was measured when the solvent surface was 6 cm above the top of the column. The flow rate was 0.55 ml/min.
• the tubing outlet was positioned 50 cm below the bottom of the column and the column was again filled with ethyl acetate.
• the flow rate was 2.3 ml/min.
• the dead volume of the column was 4.7 ml and the empty volume on top of the column was 20 ml, i.e. >4x the dead volume of the column.
• a column similar to the one in example 1 was used to separate a 1:1 mixture (total 30 mg) of N-methyl-4-nitro- aniline (R f 0.62) and 4-methoxy-2-nitroaniline (R f 0.42).
• a loop of the teflon tubing was positioned higher than the top of the column reservoir. 3 ml of chloroform was added to the column. The sample mixture dissolved in 0.3 ml of chloroform was added followed by 0.5 ml of chloroform. The column was filled with chloroform. The chromatography was started by lowering the tubing loop and positioning the tubing outlet 50 cm below the bottom of the column.
• Example 3 Three polypropylene reservoirs (25 ml, inner diameter 20 mm) were equipped with polyethylene filters and packed with 3. Og of silica of different particle size, 6-35 ⁇ , 20-45 ⁇ and 40-63 ⁇ respectively (all products from Grace Davison) . To the bottom of each column was connect- ed a valve and 50 cm of teflon tubing (inner diameter
• a large polyethylene reservoir (150 ml, inner diameter 38 mm) was equipped with a polyethylene filter and packed with 20 g of silica (6-35 ⁇ from Grace Davison con- taining 4% fluorescence indicator) and another filter placed on top.
• the height of silica was 37 mm.
• the tubing outlet was positioned 70 cm below the bottom of the column and the column was again filled with ethyl acetate. Flow rate 5.6 ml/min. The empty volume on top of the column was estimated to be 3.7x the dead volume of the column.
• a column similar to the one in example 1 was used to examine how a coloured band of compound broadened after having been applied to the column and awaiting the start of the separation.
• 3 ml of chloroform was added to the column followed by 40 mg of N-methyl-4-nitroaniline dissolved in 0.3 ml of chloroform.
• the sample was washed down with 0.5 ml of solvent, the column filled with chloroform and the solvent was allowed to reach the equillibrium point in the teflon tubing ( ⁇ 2.5 min) .

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• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Treatment Of Liquids With Adsorbents In General (AREA)

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Cited By (2)

Citations (3)

• 2001
  • 2001-12-21 SE SE0104412A patent/SE0104412D0/sv unknown
• 2002
  • 2002-12-20 WO PCT/SE2002/002430 patent/WO2003061806A1/en active Application Filing

Patent Citations (3)

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