DE10119419A1 - Repetitive preparative chromatography procedure - Google Patents

Abstract

The invention relates to a method and device for carrying out automatic, repetitive column chromatography according to which steps are repeatedly carried out in succession and at fixed points in time in each elution cycle. These steps include: a) rinsing the column; b) conditioning the column to a ballistic state; c) supplying the sample; d) eluting, and; e) a detector signal of a chromatographic measurement of the eluate is recorded, and an automatic fractionation of the eluate ensues according to the signal progression of the detector signal.

Description

The invention relates to a method for automatic repetitive preparative Chromatography.

The preparative chromatography is because of its reliability, good handling Ability and high productivity have been an important technology for cleaning for many years supply of chemical, pharmaceutical and biological preparations. She becomes one set in drug discovery, for natural product isolation, for cleaning Synthesis intermediates and chemical-pharmaceutical products, for isolation tion of the secondary components in an active ingredient or for the separation of isomers. In the repetitive mode of operation, a fractionation is repeated many times and the eluate is withdrawn at predetermined times. A repetitive way of working enables larger quantities of substance in the "one shot method" semi-preparative scale (mg to hectograms) by multiple successive performed separations of small portions of the sample under laboratory conditions clean.

Repetitive preparative chromatography has the problem of failure separation or incorrect fractionation if the fractionation cycle is in the column compared to the previously determined times, whereby this Problem due to the large number of cycles over a long period of time can keep getting worse. The problem of mismatch or misfraction Up to now, it could only be avoided by means of personnel-intensive monitoring become.

The task is therefore to prepare a method for repetitive chromatography that works automatically and error-free over long periods of time.  

The inventive method for automatic repetitive column chromatography is that the steps are repeated at fixed times in each elution cycle

• a) rinsing the column,
• b) conditioning the column to a ballistic state,
• c) the sample application
• d) Elution

be carried out and

• a) a detector signal of a chromatographic measurement of the eluate is recorded and after the waveform of the detector signal an automatic frak tion of the eluate takes place.

In repetitive chromatography using the method according to the invention worked under non-stationary conditions, i.e. the column is placed in front of one Sample application not in the state of equilibrium, but in a strictly reproducible measurable imbalance state, the so-called ballistic state, ge introduced. This applies to the use of both normal and reverse phases.

Each elution cycle begins with a short rinse of the column, preferably with a strong eluent to protect the column from highly retarding contaminants clean and at the same time bring the column into an inactive initial state. The amount of rinsing liquid is in the range of 0.1 to 3, preferably 0.5-1 des Column volume and contains a high proportion of strongly eluting solvent.

After the rinsing step, the column is briefly conditioned, preferably with a weak initial eluent to make it a reproducible initial state that is an imbalance or ballistic state. After conditioning with 0.1 to 3, preferably 0.5-1 column volumes of initial  The column is in a reproducible initial state, and the eluent follows Sample application.

The sample application and elution take place in a ballistic state.

When applying the sample, the sample is added to the column in portions. The Sample can be injected into the eluent stream reduced during the injection.

The sample amount is preferably set so that the column is overloaded. The that is, large amounts of sample are injected that span a wide zone in the Distribute column and in the chromatograms to wide and asymmetrical Peaks (substance zones) lead because the separation in the non-linear range of Adsorption isotherm takes place. This way of working makes sense as long as the desired substance peaks in the chromatograms from other sub have punching peaks separated. The peak shape in the chromatograms corresponds to that for the preparative working method typical form with plateau, fronting and tailing.

The chromatography is either isocratic, that is, it is constant with an eluent Composition throughout the chromatographic process or below a gradient with an eluent in a time-varying combination setting.

The ballistic condition of the column as a prerequisite for reproducible chromato grams and fractionation is only achieved if steps a) to e) (rinsing, Conditioning, sample application, elution and fractionation) a set, yourself form a constantly repeating cycle. They are already in the second cycle Conditions for reproducible separation and fractionation are given.

Steps a) to e) are repeated automatically until the entire sample amount is chromatographed. Fractionation always takes place during the repetition cycles in the same vessels.  

Significant eluent and time savings can be achieved if the next elution cycle is started during step e) of the previous one Elution cycle is still being processed, i.e. while the analog detector signal a chromatographic measurement of the eluate from the previous elution cycle is recorded and the automatic fractionation of this eluate takes place. This optimization is limited by the elution of the "rinsing peak" from step a) not with the last peak of interest from the previous elution cycle may coincide, but must be eluted shortly thereafter.

Under certain conditions, e.g. B. only contaminants from the samples must be removed, can be carried out in an elution cycle after rinsing step a) and conditioning b) several times the steps sample application c) and elution d) are carried out in succession, the chromatography being carried out isocratically.

This method leads to satisfactory under the conditions mentioned above Results and saves time and eluent.

The fractionation can be carried out in such a way that the eluate is in one or more specified periods in each elution cycle (collection time windows) and during the detector signal is beyond a predetermined value corresponding collecting time window is collected. collection The time window and the collecting vessel are therefore in a fixed relationship to one another.

The eluate from several collection time windows, initially in separate, the Collection time windows assigned, collection vessels can be collected be merged later.

The predetermined value can also change over time Change collection time window.  

A time shift of the collection time window in the next elution cycle by the amount of time in the previous outgoing elution cycle, the time at which the detector signal in a control time window (spacer) a certain value over or has fallen below.

The procedure can be ended automatically if the Detector signal one of several at a given point in time in the cycle predetermined control values reached. By selecting these control values a very variable, application-specific definition of the error and variabili tolerance allows. With the help of the control values, numerous security can be achieved Define criteria based on the time course and the height of the detector signals are related such as the presence of certain peaks in the Chromatogram, reaching the baseline between two substance zones, height the baseline at the start of the chromatogram etc.

The chromatographic measurement of the eluate can be carried out using standard methods such as UV VIS (visible light and UV), RI (refractive index detection), MS (masses spectroscopy) or via light scattering detection, UV-VIS and RI.

The method according to the invention is preferred with a robust and long-lasting chromatographic column performed. A column is preferably used which has a device that compresses the packing material in the axial direction (see e.g. US 5,893,971). This eliminates the risk of channel formation in the pillars medium eliminated. It can be used, for example, with silica-based Column packs hundreds of elution cycles without deterioration of the columns perform properties.

The invention further relates to a system for carrying out the inventions method according to the invention. The system includes a chromatographic column, a  System for feeding eluents and samples into this chromatographic column, a fraction collector with one or more collection vessels, a detector and a digital signal processing system with processor, control unit, Input unit, display unit and storage unit. The digital signal processing The system is controlled by a program so that the detector measured chromatograms stored in the storage unit and in the Display unit are displayed. The values entered via the input unit (Time and detector signal level) of the collection time windows are together with the associated collection vessel numbers stored in the storage unit. The values the collection time window can with the chromatograms in the display unit are displayed.

The processor compares each detector signal that is measured by the detector, with the saved values of the collection time window and in the event that the ge measure value is beyond a value specified by the collection time window, the processor then sends the signal to the control unit to the fraction collector control that the eluate is collected in the collection vessel whose Collection vessel number with this collection time window in the storage unit was saved.

Values for control time windows (time and detector signal level) can be entered and saved in the memory unit can be displayed with the measured chromatograms in the display unit can be. The processor compares each detector signal that goes through the detector is measured with the stored values of the control time window and for the Case that the time has shifted when the detector signal in the Control time window has exceeded or fallen below a certain value, the saved time values for the collection time window by the corresponding the Time period changed automatically and the changed time values of the collection time window stored in the storage unit.  

Control values can be entered via the input unit Storage unit can be saved and with the measured chromato programs can be displayed in the display unit. The processor compares each detector signal that is measured by the detector with the stored Control values and in the event that there is a match between detector signal and control value, the processor can abort the measurement.

The method according to the invention can be used for all preparative, chromatographic Separation tasks are used. It is particularly recommended for synthetic applications Areas of application in which work is carried out under a tight schedule and the Preparations are extremely valuable because of the work already invested in them and mismatches or fractionations are intolerable.

The advantage of the method according to the invention is also that larger amounts of sample can be cleaned with comparatively small columns. So can also be Use extremely costly phases that are not for large columns due to cost reasons be available. Malfunctions and incorrect fractionations are avoided without the need for personnel-intensive monitoring and valuable samples are at risk. With the method according to the invention, eluent and time can be determined save up.

Figures and examples

The figures show

Fig. 1 sequence of a repetitive chromatography.

Fig. 2 chromatograms with collection time windows, control time window (from stand) and control values (control lines).

Fig. 3a chromatogram in the removal of a polar contamination with collection time window and control values.

FIG. 3b, the end of the repetitive chromatography in the removal of a polar impurity.

example 1

Fig. 1 shows an example of the flow of the process of the invention. The diagram below shows the solvent composition in% plotted over time. The two eluents A 13 and B 12 were applied to the column over a period of 10 min per cycle. A sample injection takes place at the time of the markings 11 . The upper diagram shows the chromatogram obtained.

The sequence of a cycle of repetitive chromatography is now as follows: At the beginning of the cycle, the column is rinsed for 1 min with 100% eluent A 13 (rinsing phase 16 ). The associated rinsing peak 14 can be seen in the chromatogram. The conditioning is then carried out for 2 minutes, in which the ratio of A 13 to B 12 is 80% to 20%. Now the sample task follows. During the remainder of the cycle, the proportion of eluent A 13 increases to 90% and the proportion of eluent B 12 decreases accordingly. About 4 to 5 minutes after the sample application (or 7 to 8 minutes after the start of the cycle), the first characteristic peaks of the chromatogram are measured and the eluate is fractionated accordingly.

The chromatogram 10 shown in FIG. 1 was achieved with a self-made column made of Hyperprep HS C18 - 8 μm with the column dimensions (length × diameter) 24 cm × 2 cm. Up to a grain size of 8 µm, the column can still be easily filled by hand using a vacuum. A slurry pump was used for smaller grain sizes. The eluent system consisted of three Gilson HPLC pumps 30 x with manometer module and mixing chamber. The sample pump was a Gilson 30 x, the detector was a Knauer UV monitor at 210, the fraction collector was a Gilson 206 . Methanol was used as eluent A and H ₂ O was used as eluent B. The sample consisted of the alkylbenzenes C1, C3, C5 with 1 g / 100 ml each in ACN, to which the alkylbenzenes C5, C6, C7 each with 0.2 / 100 ml and 1.5 ml acetone were added. The injection speed of the sample was 11.2 ml / min and the eluent flow was 20 ml / min.

Example 2

FIG. 2 shows an example of how the collection time windows 1 , 2 , 3 , 4 , control time window 24 and control values 25 were determined in the chromatograms ( 21 , 22 , 23 ). First of all, pre-optimization of the elution conditions was carried out using small amounts of sample. For pre-optimization, different gradients were run in sequence with the same sample. The separation conditions were thus determined.

After sufficient separation and baseline stability was achieved, the collection time windows 1 , 2 , 3 , 4 were determined for the fractionation.

On the basis of several superimposed test chromatograms 21 , 22 , 23 (cycle no. 13, 50 and 150) it can be seen that the chromatograms do not differ significantly from one another.

The collection time windows 1 , 2 , 3 , 4 define the start and end of the fractionation into a specific collection vessel. The position of the collection time windows 1 , 2 , 3 , 4 with respect to the detector signal values defines the detector signal level from which the fractionation takes place within the corresponding collection time window. In this way it is precisely defined which eluate parts are collected.

The collection time windows can overlap in time, as shown in FIG. 2 for the collection time windows 1 and 2 or 3 and 4 . In such a case, it is stipulated which collection time window takes precedence in the overlap period, i.e. which collection vessel is fractionated in the overlap period. Preference is given to the collection time window that started earlier. It is fractionated into the vessel belonging to this collection time window until this collection time window has run through completely and then, after the overlap period, fractionation into the vessel assigned to the next collection time window only begins.

The determination of the collection time window 1 , 2 , 3 , 4 is preferably carried out using a suitable software program which allows the lines representing the collection time window 1 , 2 , 3 , 4 to be drawn directly into the chromatograms 21 , 22 , 23 , and that thus selected values for evaluating the chromatograms recorded in each eluent cycle and the corresponding control of the eluate flow into the individual vessels.

If the retention times shift in the unattended repetitive elution cycles, the fractionation is automatically adapted. A control time window 24 is determined analogously to the collection time windows 1 , 2 , 3 , 4 with respect to the associated detector signal values. The control time window 24 should contain the ascending or descending branch of a reliable reference peak in the chromatogram. The intersection of the detector signal with the control time window 24 is evaluated after each cycle in order to track the collection time windows 1 , 2 , 3 , 4, if necessary, of a shift in the retention time.

The gradient duration can also be updated if this is due to Retention time shifts should be necessary.

The control values 25 were introduced for error control. If the control values 25 are reached by the detector signal at the specified times, the fractionation is stopped immediately. The control values 25 have the effect that the ongoing fractionation process is not continued if the chromatograms shift so much in time in the course of the elution cycles that the collection time windows no longer lie in the desired range of peaks in the chromatogram.

The chromatograms ( 21 , 22 , 23 ) shown in FIG. 2 were recorded with the experimental setup described in Example 1. Methanol was used as eluent A and H ₂ O was used as eluent B. The sample consisted of 13.5 g / 90 ml of an unknown active ingredient. The injection volume was 0.7 ml. The injection rate of the sample was 11.2 ml / min and the eluent flow was 20 ml / min.

Example 3

Fig. 3b shows an example of an alternative sequence of the method according to the invention with samples that were already well pre-cleaned, and where more contaminants should only be removed. Figure 3b gives the solvent composition in% plotted against time. The two eluents C 34 and A 36 were placed on the column over a hundred cycles. A sample injection took place at the time of the markings 35 . Fig. 3a shows the chromatogram obtained.

In contrast to Example 1, the ratio of Eluents kept constant over the entire duration of the elution. In each Cycle only took place at the beginning of the rinsing step and conditioning, afterwards followed by ten sample assignments and elution in quick succession. So 90% time and eluent saved for rinsing and conditioning. The Chromato grams were continuously recorded and the eluate fractionated accordingly.

Fig. 3a shows an example of how the collection time window 32 and the control values 33 have been defined. This method is particularly suitable for separations that can be carried out isocratically. The collection time window 32 defines the start and end of the fractionation. The collection time window 32 is set so high with respect to the detector signal values that the peaks of the contaminant are safely below it. The fractionation always took place when the detector signal rose above the collection time window 32 . All peaks of the main component were collected in the same vessel. When the detector signal fell below the fractionation line, the fraction collector moved to a drop position.

The presence of a sample in the eluate is monitored by the control values 33 . If there is no longer any sample in the eluate, then the chromatogram has no peak in the area of the control values 30 and the control values 33 are reached by the detector signal and the fractionation process is stopped.

The control values 34 limit the retention time fluctuations. The fractionation process stops when one of the peaks reaches one of the control values 34 .

. The chromatogram shown in Figure 3a was treated with a home-built column of Hyperprep HS C18 - 8 microns with the column dimensions 18, 5 x 2 cm obtained. Up to a grain size of 8 µm, the column could easily be filled by hand using a vacuum; for smaller grain sizes, a slurry pump is used. The eluent system consisted of three Gilson HPLC pumps 30 x with manometer module and mixing chamber. The sample pump was a Gilson 30 x, the detector was a Knauer UV monitor at 210, the fraction collector was a Gilson 206 . H ₂ O was used as eluent A 36 and methanol was used as eluent C 34. The sample consisted of 89 g of active ingredient in 340 ml of ACN. The injection rate of the sample was 11.2 ml / min and the eluent flow was 20 ml / min. The injection volume was 0.4 ml, the pressure 65 bar.

Claims (20)

1. Method for automatic repetitive column chromatography, in which the steps are repeated successively and at fixed times in each elution cycle

• a) rinsing the column,
• b) conditioning the column to a ballistic state,
• c) sample application,
• d) elution

be carried out and

• a) a detector signal of a chromatographic measurement of the eluate is recorded and an automatic fractionation of the eluate takes place after the signal curve of the detector signal.

2. The method according to claim 1, characterized in that the flushing in Step a) with a strong eluent and conditioning the column in Step b) is carried out with a weak initial eluent. 3. The method according to claim 1 or 2, characterized in that the amount of rinsing liquid in step a) in the range from 0.1 to 3, preferably 0.5 to 1 of the column volume and the amount of the initial eluent in step b) im Range of 0.1 to 3, preferably 0.5 to 1 of the column volume. 4. The method according to any one of claims 1 to 3, characterized in that the sample quantity in the sample application in step c) is dimensioned such that the column is overloaded.   5. The method according to any one of claims 1 to 4, characterized in that the eluent composition is constant during each elution cycle. 6. The method according to any one of claims 1 to 4, characterized in that the eluent composition during each elution cycle changes a given gradient. 7. The method according to any one of claims 1 to 6, characterized in that the next elution cycle is started during step e) of the previous cycle is still being processed. 8. The method in deviation from one of claims 1 to 7, according to the rinsing step a) and the conditioning b) the steps several times Sample recording c) and elution d) are carried out in succession and the Eluent composition constant throughout the elution cycle remains. 9. The method according to any one of claims 1 to 8, characterized in that the eluate in defined collection time windows in each elution cycle and while the detector signal is beyond a predetermined value in a collecting vessel assigned to the collecting time window is collected. 10. The method according to claim 9, characterized in that at least two Overlap collection time window and enter for the overlap period Collection time window has priority and during the overlap period in the frak collection container assigned to this priority collection time window is tioned. 11. The method according to claim 9 or 10, characterized in that the predetermined value over the course of time of the collection time window changes.   12. The method according to any one of claims 9 to 11, characterized in that the eluates in separate collection containers assigned to the collection time windows collected and later merged. 13. The method according to any one of claims 9 to 12, characterized in that an automatic time shift of the collection time window in the next following cycle takes place for the amount of time in which before current elution cycle has shifted the time at which the detector signal over or over a certain value in a control time window has fallen below. 14. The method according to any one of claims 1 to 13, characterized in that the execution of the method is automatically ended when the Detector signal at a predetermined time in the elution cycle achieved by several predetermined control values. 15. The method according to any one of claims 1 to 14, characterized in that the chromatographic measurement of the eluate using UV-VIS, RI, MS or Light scatter detection, preferably carried out via UV-VIS or RT. 16. The method according to any one of claims 1 to 15, characterized in that the chromatographic column used is axially compressed. 17. System for carrying out the method according to one of claims 1 to 16, comprising a chromatographic column, a system for feeding Eluents and samples in this chromatographic column, a fraction collector with one or more collection vessels, a detector and a digital signal processing system with a processor, an input unit, a control unit, a display unit and a storage unit, characterized in that the chromatograms measured with the detector  stored in the storage unit and in the display unit are displayed and values entered via the input unit (time and Detector signal height) of the collection time window together with the associated Collecting vessel numbers are stored in the storage unit and the values the collection time window with the chromatograms in the display unit are displayed. 18. System for performing the method according to claim 17, characterized characterized that the processor any detector signal that over the The detector is measured using the stored values of the collection time window compares and in the event that the measured value is beyond one by the Collection time window predetermined value is, via the control unit Fraction collector controls so that the eluate is collected in the collecting vessel whose collection vessel number with this collection time window in the Storage unit is stored. 19. System for performing the method according to claim 17 or 18, characterized characterized in that the values of the Control time window (time and detector signal level) in the memory unit saves and with the measured chromatograms in the display unit are displayed and the processor of each detector signal, which over the Detector is measured with the stored values of the control time window compares and in the event that the time has shifted to the detector signal in the control time window exceeds a certain value or has fallen below the saved time values for the collection time window by the corresponding time span and the changed time values of the Collection time windows can be stored in the storage unit. 20. System for performing the method according to one of claims 17 to 19, characterized in that entered via the input unit Control values are stored in the storage unit and with the measured ones  Chromatograms are displayed in the display unit and the processor each detector signal that is measured by the detector with the stored control values and in the event that an over The measurement is in agreement between the detector signal and the control value aborts.