阅读说明：本技术 控制制备液相色谱的方法和系统 (Method and system for controlling preparative liquid chromatography ) 是由 莱昂内尔·博赫 迪迪埃·沙博诺 阿兰·查柏亚 多米尼克·戴思夸伊瑞 奥利维尔·梅西埃 雅恩 于 2019-04-08 设计创作，主要内容包括：本发明涉及一种用于控制制备液相色谱的方法,其包括以下步骤,所述步骤的至少一部分由包括处理器和耦合至所述处理器的显示屏的计算机来实施：(a)从薄层色谱(TLC)和高效液相色谱(HPLC)中选择分析液相色谱法,(b)输入通过步骤(a)选择的方法获得的待纯化产品的分析液相色谱数据,(c)访问用户可用的用于实施所述制备液相色谱的分离工具的表,(d)由所述分析液相色谱数据和可用分离工具的表,从所述表中选择最佳分离工具,并计算用于所述选择的分离工具的制备液相色谱操作条件。(The present invention relates to a method for controlling preparative liquid chromatography comprising the following steps, at least a part of said steps being implemented by a computer comprising a processor and a display screen coupled to said processor: (a) selecting an analytical liquid chromatography from Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC), (b) inputting analytical liquid chromatography data of the product to be purified obtained by the method selected in step (a), (c) accessing a table of separation tools available to a user for performing the preparative liquid chromatography, (d) selecting an optimal separation tool from the table from the analytical liquid chromatography data and the table of available separation tools, and calculating preparative liquid chromatography operating conditions for the selected separation tool.)

1. A method for controlling preparative liquid chromatography comprising the steps of, at least a portion of, being implemented by a computer comprising a processor and a display screen coupled to the processor:

(a) selecting analytical liquid chromatography from Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC),

(b) inputting analytical liquid chromatography data of the product to be purified obtained by the method selected in step (a),

(c) accessing a table of separation tools available to a user for performing the preparative liquid chromatography,

(d) from the analytical liquid chromatography data and a table of available separation tools, an optimal separation tool is selected from the table, and preparative liquid chromatography operating conditions for the selected separation tool are calculated.

2. The method of claim 1, further comprising the step of inputting the amount of product to be purified.

3. The method according to one of claims 1 or 2, further comprising the step of selecting the manner of introducing the product to be purified into the selected separation means, said manner of introduction being selected from introduction in solution or solid deposition.

4. A method according to one of claims 1 to 3, comprising a step of verifying the calculation by the user, and, after said verification, a step of initiating preparative liquid chromatography on the selected separation tool.

5. The method of one of claims 1 to 4, comprising triggering by a user an acceleration of the preparative liquid chromatography, step (d) comprising calculating optimal operating conditions while taking into account the maximum flow rate and the limiting pressure of the preparative liquid chromatography system implementing the selected separation tool.

6. The method according to one of claims 1 to 5, wherein the method selected in step (a) is Thin Layer Chromatography (TLC) and step (b) comprises the input of analytical data obtained on a thin layer chromatography plate.

7. The method of claim 6, wherein the selection and calculation carried out in step (d) takes into account four different cases according to the retention factor (Rf) value of the plate:

-0.01<Rf<0.07，

-0.08<Rf<0.4，

-0.4<Rf<0.9，

-0.9<Rf<1.0。

8. the method of claim 7, comprising adapting the shape of the elution gradient to each value of a volume difference factor (Δ CV) while taking into account the fact that the same factor (Δ CV) corresponds to a difference (Δ Rf) in different retention factors in each of the four regions.

9. The method according to one of claims 6 to 8, wherein the selection and calculation carried out in step (d) takes into account one to all of the compounds of interest spread over the entire plate.

10. The method according to one of claims 6 to 9, wherein step (d) takes into account the difference (Δ ∈) between the elution forces of the solvents used in preparative liquid chromatography and the non-linear variation of the elution force of the solvent mixture with the composition of the mixture.

11. A method according to any one of claims 6 to 10, comprising acquiring an image of a thin layer chromatography plate and automatically detecting the separated compounds from the image by a user's portable device, the inputting of analytical data of step (b) comprising importing data from the portable device.

12. The method of any one of claims 1 to 5, wherein the method selected in step (a) is high performance liquid chromatography, said selecting further comprising selecting a chromatography mode from the group consisting of:

normal Phase Liquid Chromatography (NPLC),

-Reverse Phase Liquid Chromatography (RPLC),

-hydrophilic interaction chromatography (HILIC),

-Hydrophobic Interaction Chromatography (HIC).

13. The method of claim 12, wherein the analytical chromatographic data input in step (b) is analytical liquid chromatographic data obtained on a reference separation tool.

14. The method of claim 13, wherein the list of available separation tools comprises the same separation tools as the reference separation tool, and step (c) comprises selection of the tools and calculation of preparative liquid chromatography operating conditions for the separation tools.

15. The method according to claim 13, wherein the table of available separation tools does not include the same separation tool as the reference separation tool, and step (d) comprises selecting a separation tool having a stationary phase different from the stationary phase of the reference tool by comparing similarities between the stationary phase of the reference tool and the stationary phase of the available separation tool, or selecting a separation tool comprising the same stationary phase as the reference tool and calculating preparative liquid chromatography operating conditions in multiple sample injection mode while minimizing the number of sample injections.

16. The method according to one of claims 6 to 15, comprising the step of calculating the degree of separation by thin layer chromatography or by high performance liquid chromatography under isocratic elution to determine further analytical conditions.

17. The method according to one of claims 6 to 16, wherein the selection and calculation of step (d) takes into account the elution forces specific to each strong solvent.

18. The method according to one of claims 1 to 4, comprising performing a separation test on a plurality of separation tools and selecting the separation tool with the best separation potential from the tools.

19. The method of claim 18, wherein the list of available separation tools includes the same separation tool as the tool with the best separation potential, and step (d) includes selection of the tool and calculation of preparative liquid chromatography operating conditions for the separation tool.

20. The method of claim 18, wherein the list of available separation tools does not contain the same separation tool as the tool with the best separation potential, and step (d) comprises selecting a separation tool that contains the same stationary phase as the tool with the best separation potential and computationally preparing liquid chromatography operating conditions in multiple sample mode while minimizing sample injection times.

21. The method according to one of claims 1 to 20, comprising ordering at least two series of steps (a) to (d) with two different analytical liquid chromatography methods and/or modes.

22. The method of claim 21, wherein the first series of steps (a) through (d) is performed automatically when the first series of steps (a) through (d) does not identify the preparation of the liquid chromatography solution.

23. A preparative liquid chromatography system comprising:

a computer comprising a processor, a user interface and a display screen, the processor being configured to implement the steps of the method according to one of claims 1 to 22,

-a preparative liquid chromatography system controlled by the computer comprising a mobile phase tank, a pump, a sample injector, a separation means, a detector, a data logger and a fraction collector.

24. Computer program product comprising program code instructions recorded on a computer readable carrier, characterized in that it comprises instructions for implementing a method according to one of claims 1 to 22.

Technical Field

The present invention relates to a method and system for the controlled preparation of liquid chromatography from low pressure to high pressure.

Background

Preparative liquid chromatography is a method for separating, purifying or enriching a mixture of compounds, which for optimization has to be selected a certain number of parameters (internal diameter, length, stationary phase, particle size, etc.) and operating conditions (mobile phase, elution type, flow rate, temperature, mode of injection, amount of sample injection, etc.) with respect to the separation tool used. The selection of these parameters is generally attributed to a productivity compromise between the desired purity level, the amount of pure product required, the cost and duration of the purification process.

To make this choice, it is known to use the separation results obtained for samples having the same properties with an analytical chromatographic system and transpose it to a preparative liquid chromatography. Thus, the term "scale-up" defines the multiplier factor between the amount of sample processed in analytical liquid chromatography and the amount of sample processed in preparative liquid chromatography.

However, analytical liquid chromatography systems are very different from preparative liquid chromatography systems in terms of stationary phase and operating conditions, and therefore such a transposition is relatively complicated. Accordingly, computational tools have been designed to facilitate such transpositions.

Thus, document US 7,686,959 describes a computer-implemented method of calculating preparative chromatographic operating conditions using Thin Layer Chromatography (TLC) data. More specifically, this document teaches the calculation of the pre-ratio R of two compounds of a sample to be purified by thin layer chromatography_f1、R_f2Calculating the column volume CV for each of said compounds from their respective pre-ratios₁、CV₂(each column volume equals the reciprocal of the respective previous ratio), and the calculation is defined as the volume CV₂And CV₁The column factor Δ CV of the difference. However, this factor Δ CV is specific to each column, which requires a large number of calculations to determine the best column. Furthermore, this method requires a lot of intervention (data input) by the user, which assumes that the user has reliable chromatographic knowledge.

However, it would be useful to provide a control method and system that is more highly automated and requires minimal operator intervention, as far as the operator responsible for performing preparative liquid chromatography purification, not necessarily a chromatography specialist, is concerned.

Disclosure of Invention

It is therefore an object of the present invention to devise a method of controlling preparative liquid chromatography which can be performed by novices at the chromatographic side while minimizing the amount of data entered manually, thereby determining the optimal and sufficient separation tools and purification conditions as a function of the expected results.

To this end, the invention proposes a method for controlling preparative liquid chromatography comprising the following steps, at least part of said steps being carried out by a computer comprising a processor and a display screen coupled to said processor:

(a) selecting analytical liquid chromatography method from Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC),

(b) inputting analytical liquid chromatography data of the product to be purified obtained by the method selected in step (a),

(c) accessing a table of separation tools available to a user for performing the preparative liquid chromatography,

(d) from the analytical liquid chromatography data and a table of available separation tools, an optimal separation tool is selected from the table, and preparative liquid chromatography operating conditions for the selected separation tool are calculated. This step comprises, inter alia, determining a suitable gradient profile.

By "separation means" is meant a column, cartridge or any other chromatographic device comprising a stationary phase.

The method further comprises the step of receiving an amount of product to be purified.

According to one embodiment, the method further comprises the step of selecting the way in which the product to be purified is introduced into the selected separation means, said introduction being selected from introduction in solution or solid deposition.

Advantageously, the method further comprises a step of validating the calculation by the user, and, after said validation, a step of initiating a preparative liquid chromatography on the selected separation tool.

According to one embodiment, the method comprises triggering the acceleration of the preparative liquid chromatography by a user. Step (d) then comprises calculating optimal operating conditions while taking into account the maximum flow rate and the limiting pressure of the preparative liquid chromatography system implementing the selected separation tool.

According to one embodiment, the method selected in step (a) is Thin Layer Chromatography (TLC). Step (b) then comprises the input of analytical data obtained on the TLC plate.

Preferably, the selection and calculation carried out in step (d) takes into account four different cases of retention factor Rf values according to the panel:

-0.01<Rf<0.07，

-0.08<Rf<0.4，

-0.4< Rf <0.9, and

-0.9<Rf<1.0。

in a particularly advantageous manner, the method comprises adapting the shape of the elution gradient to each value of the volume difference factor Δ CV, while taking into account the fact that the same factor Δ CV corresponds to a different retention factor difference Δ Rf in each of the four regions.

Preferably, the selection and calculation carried out in step (d) takes into account one to all of the compounds of interest spread over the entire plate.

In an advantageous manner, step (d) takes into account the difference between the elution forces of the solvents used in preparative liquid chromatography and the non-linear variation of the elution force of the solvent mixture with the composition of said mixture.

According to one embodiment, the method comprises acquiring an image of a thin layer chromatography plate and automatically detecting the separated compounds from the image by a user's portable device, the inputting of analytical data of step (b) comprising importing data from the portable device.

According to one embodiment, the method of step (a) is selected from high performance liquid chromatography, said selecting further comprising selecting the chromatography mode from:

-Normal Phase Liquid Chromatography (NPLC),

-Reverse Phase Liquid Chromatography (RPLC),

-hydrophilic interaction chromatography (HILIC),

-Hydrophobic Interaction Chromatography (HIC).

The analytical chromatographic data input in step (b) is then the analytical liquid chromatographic data obtained on the reference separation tool.

According to one embodiment, the table of available separation tools comprises the same separation tools as the reference separation tool, and step (d) comprises selection of said tools and calculation of preparative liquid chromatography operating conditions for said separation tools.

Alternatively, the table of available separation tools does not include the same separation tool as the reference separation tool, and step (c) includes selecting a separation tool having a stationary phase different from that of the reference tool by comparing similarities between the stationary phase of the reference tool and the stationary phase of the available separation tool, or selecting a separation tool having the same stationary phase as the reference tool and calculating preparative liquid chromatography operating conditions in a multiple sample (or multiple run) mode while minimizing sample injection times.

According to one embodiment, the method comprises the step of calculating the degree of separation by thin layer chromatography or by high performance liquid chromatography under isocratic elution (iso-elution) to determine further analytical conditions.

In an advantageous manner, the selection and calculation of step (d) takes into account the elution forces specific to each strong solvent.

According to one embodiment, the method comprises conducting a sieving separation test on a plurality of separation tools and selecting the separation tool with the best separation potential from the tools. This test is performed in the absence of analytical data in step (b) which is easily entered, whether in thin layer chromatography or in high performance liquid chromatography.

According to one embodiment, the table of available separation tools comprises the same separation tool as the tool having the best separation potential, and step (d) comprises selection of said tool and calculation of preparative liquid chromatography operating conditions for said separation tool.

In an alternative, the table of available separation tools does not include the same separation tool as the tool having the best separation potential, and step (d) includes selecting a separation tool that includes the same stationary phase as the tool having the best separation potential, and calculating preparative liquid chromatography operating conditions in multiple sample injection mode while minimizing sample injection times.

According to one embodiment of the invention, the method comprises ordering at least two series of steps (a) to (d) with two different analytical liquid chromatography methods and/or modes.

In a particularly advantageous manner, the sequencing is carried out automatically when the first series of steps (a) to (d) does not identify the preparation of a liquid chromatography solution.

Another object of the invention is a system for implementing said method.

The system comprises:

a computer comprising a processor, a user interface and a display screen, the processor being configured to implement the steps of the method, for example, as described above,

-a preparative liquid chromatography system controlled by the computer comprising a mobile phase tank, a pump, a sample injector, a separation means, a detector, a data logger and a fraction collector.

Preferably, the control method described above will be implemented by a processing device comprising means for implementing the steps of the control method, for example a PC type computer comprising a memory and a processing unit on which a computer program is executed.

The computer program comprises, inter alia, one or more algorithms capable of performing the steps of the method described above.

Drawings

Other features and advantages of the present invention will become apparent from the following detailed description, which, when taken in conjunction with the drawings,

wherein:

FIG. 1 is a logic diagram illustrating the operation of a system according to the present invention;

FIG. 2 is a separation profile obtained on a TLC plate.

Detailed Description

The invention enables the best possible purification compromise to be presented by analytical chromatographic analysis, in particular on thin-layer or HPLC (high performance liquid chromatography or high pressure liquid chromatography) columns, in normal phase, reversed phase or otherwise, by preparative liquid chromatography.

The preparative liquid chromatography can be carried out from low to high pressure and includes in particular the names: flash chromatography, MPLC (Medium pressure liquid chromatography) and HPLC. The implementation pressure is only limited by the chromatography system used, in particular the pressure that the separation tool can withstand. "Low pressure" generally means a pressure of about a few bar to a few tens of bar; "high pressure" generally refers to pressures of several tens to several hundreds of bars, or even higher.

To this end, the present invention proposes a computer-implemented method that guides the user from providing analytical data up to defining all the conditions under which preparative liquid chromatography may be performed. All steps may be performed automatically without prompting the user to enter data or make a selection.

This method is therefore of particular interest to novices in the field of chromatography.

However, even if the user has a certain level of chromatographic expertise, the method is still advantageous in that it can reduce the time required to determine the operating conditions; furthermore, the method may comprise steps directly implemented by the user, which enable the user to interact with the algorithm used (expert mode).

The method includes accessing a table of separation tools available to a user. This table may be in the form of a database listing all tools in the user's inventory and including fields corresponding to the primary features of the tools: the type of tool (e.g., column, barrel), the material of which the tool is constructed (plastic, stainless steel, etc.), the dimensions (inner diameter, width, etc.), the nature of the stationary phase, the particle size of the stationary phase, the manufacturer or supplier of the stationary phase, etc. In the rest of the text, the term "column" will be used for the sake of brevity, but there is no limitation as to the type of separation tool used. In a particularly advantageous manner, the preparation means make it possible to carry out different chromatographic techniques, in particular techniques that can be carried out at different pressures (from low to high pressure), in normal and/or reverse phase.

The process can advantageously be carried out by the Applicant under the trade name puriFlash^TMThe chromatographic system is sold for implementation, but may be implemented in other systems. These systems typically include a mobile phase tank, a pump, an injector, a separation tool, a detector, a data logger, and a fraction collector. Such systems are well known to those skilled in the art and will therefore not be described in more detail herein.

The method may include accessing a table of chromatography systems (or devices) available to a user. The method takes into account the requirements expressed by the user according to the stage of product development, for example in terms of the purity achieved, to propose the most suitable chromatography system. The system is defined taking into account the hold-up volume of the device and the necessary pumping power. Thus, the method allows the selection of the most suitable chromatographic tools and systems depending on the product to be purified and the purity desired.

The control unit includes a processor configured to implement a number of the algorithms described below, or to communicate with a remote computer implementing the algorithms. Optionally, in the case of a solid sample, the system may also include a solid sample carrier (often referred to as dry loading); the carrier is arranged on the mobile phase loop upstream of the column.

The logic diagram of fig. 1 illustrates the general architecture of the method.

The different modalities are described in turn below.

Interestingly, all modalities are ordered such that they can optionally be implemented entirely by TLC analysis data, which is the most commonly available data in the user's inventory, while other types of analysis data are employed when TLC analysis data is not available or satisfactory. Thus, when one type of analytical data fails to provide a user with a preparative liquid chromatography protocol, the user will be automatically guided to another type of analytical data that can ultimately present a separate liquid chromatography protocol. Thus, even if he is a novice hand in chromatography, the user will be guided entirely from providing analytical data to obtaining a preparative liquid chromatography protocol.

Naturally, depending on the nature of the sample (polar or non-polar) and the nature of the available analysis data, only some of these steps may be performed, the user being free to initiate the implementation of the method from any of the modalities. However, the system is configured to be able to process all types of analytical data as detailed below.

In a first step, a user is prompted via an interface to select an analytical liquid chromatography method to be performed to obtain analytical data to be used. Selecting from the following methods, the methods suggested to the user through the interface:

-Thin Layer Chromatography (TLC),

-High Performance Liquid Chromatography (HPLC).

The selection also includes, if appropriate, the chromatographic profile used to obtain the data on the HPLC column. The chromatographic pattern is typically selected from:

normal Phase Liquid Chromatography (NPLC),

-Reverse Phase Liquid Chromatography (RPLC),

-hydrophilic interaction chromatography (HILIC): this technique is particularly advantageous for the separation of small polar molecules.

-Hydrophobic Interaction Chromatography (HIC): this technique is particularly advantageous for the separation of hydrophobic amino acids, polypeptides and proteins.

In a second step, the analysis data obtained by the previously selected method is provided as input data to the processor via the interface. As will be described in detail below, this data may be manually entered by the user in a table or imported from a portable device (e.g., a smartphone or tablet) in which the data is stored.

The first series of cases (P) relates to polar samples.

According to a first possibility (block 1), the user obtains the analytical data of the sample under consideration on available TLC plates.

The manipulation of TLC analysis data has been the primary goal of the inventors.

First, review the definition of the amounts used:

retention factor Rf is the relative amount characterizing the elution of a compound on a Thin Layer Chromatography (TLC) plate. It is the ratio between the migration distance of the solute and the migration distance of the mobile phase. Thus, the retention factor of a fully eluted solute is equal to 1. The retention factor of solutes not carried by the mobile phase is equal to 0.

-k is a thermodynamic quantity characterizing the solute retention in the column, equal to (tr-t0)/t0 or (Vr-V0)/V0, where tr is the retention time of the solute, t0 is the time required for the mobile phase to pass through the column, Vr is the volume of the mobile phase required to absorb the solute from the column, and V0 is the pore volume of the column.

For the same chromatographic system, there is a fundamental relationship that links the retention factors Rf and k:

k＝(1-Rf)/Rf

-ak is a relative quantity independent of the column geometry, which is a characteristic of a given solute eluted by a given mobile phase on a given stationary phase at a given temperature.

In preparative liquid chromatography, the volume of mobile phase required to collect the solute is a truly useful amount. This volume Vs, sometimes referred to as CV, is expressed in units of the hold-up volume of the column to account for different column geometries:

Vs＝CV＝Vr/V0＝1+k。

fig. 2 shows in a schematic way a TLC plate with two spots, each corresponding to a compound a, b, one of which is the compound of interest and the other is its closest neighbor (neighbour). d_sIs the distance between the deposited line of the sample (denoted by the letter D) and the solvent front (denoted by the letter F). d_aIs the distance between the sample deposition line and the center of the spot corresponding to compound a, d_bIs the distance between the sample deposition line and the center of the spot corresponding to compound b.

Rf_a＝d_a/d_s

Rf_b＝d_b/d_s

Vs_a＝CV_a＝1/Rf_a＝1+k_a

Vs_b＝CV_b＝1/Rf_b＝1+k_b

Thus: Δ CV ═ CV_b-CV_a

k＝K_trX (1/Rf-1), wherein K_trConstant 1

Δk＝K_tr×[(1/Rf_b-1)–(1/Rf_a-1)]

Thus: Δ CV ═ Δ k

The invention can be practiced with TLC plates having a retention factor Rf of between 0.01 and 1.0 for a compound of interest, a retention difference Δ k between the compound of interest and its nearest neighbor (or its two nearest neighbors) of greater than or equal to 0.20 and Δ Rf ≧ 0.02.

One to all compounds present on the TLC plate that are capable of spreading across the entire TLC plate may be considered. Thus, a transposition from thin layer chromatography to preparative liquid chromatography is always possible, which makes it possible to propose a purification scheme to the user without additional manipulation by the user.

The originality of this process is that it takes into account the amplitude Δ ε ° (that is to say the difference between the elution forces of the two solvents carried out alone) and the non-linear variation of the elution force of the pure solvent mixture with the composition of the mixture, ε ° being the elution force of the solvent.

Five working amplitudes are calculated which represent all the amplitudes covering the solvent elution force entered in the software.

In addition, the inventors have defined four retention zones for compounds of interest based on their absolute and relative positions on the TLC plate.

For example, for the paired cyclohexane/ethyl acetate (Δ ∈ 0.38), the following regions are defined:

(0.01<Rf≤0.07)-(0.08≤Rf≤0.40)-(0.40<Rf≤0.9)-(0.9<Rf<1.0)

also, for each of these regions, the following six strong solvent conditions have been defined to gradually simulate the curvature of the elution force (strong solvent is the easiest eluting solvent in the mobile phase):

(0 <% strong S.ltoreq.4) - (5. ltoreq. strong S.ltoreq.8) - (9. ltoreq. strong S.ltoreq.13) - (14. ltoreq. strong S.ltoreq.20) - (21. ltoreq. strong S.ltoreq.53) - (54. ltoreq. strong S.ltoreq.100),

where% strong S is the volume percentage of strong solvent in the mobile phase.

For each of these retention zones, the calculation of Δ k of the key pair or triplet (that is, the worst resolved pair or triplet of peaks) makes it possible to propose a certain number of gradient conditions and isocratic conditions when the pore volume V0 of the column is known, as a function of the column particle size (block 4).

This resulted in a total of 1215 cases (for 115 of these cases, separation was not possible).

The shape of the elution gradient is adapted to each value of Δ CV while taking into account the fact that the same factor Δ CV corresponds to the difference Δ Rf of the different retention factors in each of the aforementioned four retention zones.

From these elements, a computer algorithm has been constructed to rank all conditions relative to the four regions and generate a unique preparative liquid chromatography protocol. The algorithm integrates the calculation of a direct amplification factor based on the size of the column to be used depending on the amount of sample to be purified. The factor is a scale-up factor between the amount of material in analytical chromatography and preparative liquid chromatography.

The user indicates the quality of the sample to be purified as a supplementary input data.

The user can also select the mode of introduction of the product to be purified from the introduction in solution or solid deposition (dry load).

An algorithm calculates the best correlation between the geometry and properties of a chromatography column based on a load equation, thereby improving the cost/productivity ratio of preparative liquid chromatography.

Initial conditions and gradient shape were calculated from Rf and Δ Rf of solutes in TLC. The proposed different gradient curves are based on the shape of the elution force of binary mixtures of solvents in HPLC with normal phase polarity (normal phase mode).

The classification of the six columns is ordered according to the separation difficulty Δ k and Δ Rf (box 6). This classification is done from a table of columns, so that the proposal contains at least three columns in the user's inventory. This classification takes into account the type of column, the type of stationary phase, and the molecular weight of the compound of interest.

Thus, the display of suggested columns from all available columns is adjusted according to the target application and the input data.

The user is advantageously asked to verify the selected column (box 7).

There are different ways of inputting analytical data.

According to one embodiment, the user manually enters the analysis data into the table to completion (block 2). Data entry typically includes:

for TLC: rf, the compound of interest, the solvent and the mass to be purified;

-for HPLC: retention time of the compound, sum of the hold up volumes upstream of the column, internal diameter of the column and its height, particle size of the stationary phase, hold up volume of the analytical column, flow rate and volume of the feed.

According to a particularly advantageous embodiment, the user has at his disposal a smart portable device (e.g. a smartphone or tablet) equipped with an application for the acquisition of TLC analysis data and the transmission of said data to a processor for elaborating the preparative liquid chromatography protocol (block 3). Such an application has the advantage of making the thin layer chromatography step and its processing faster and simpler.

The application program has the following functions:

(1) automated detection of compounds on TLC plates:

the TLC plate pictures taken directly by the user or downloaded from a library of pictures located in the user's smartphone, the algorithm outlines the shape (if contrast allows) and places a dot in its center to indicate the presence of the compound. If the algorithm does not automatically identify the compound, the user may manually identify the compound or make a correction. He must also place two lines representing the sample deposition line and the solvent front.

(2) Compounds of interest were identified and retention factors (Rf) and Δ CV (═ Δ k) were calculated

Once all compounds are identified, the user indicates the compound of interest by clicking on them. Retention factor Rf is shown for each compound of interest. If the user wishes, he can check whether his compound is in the appropriate work area by pressing a dedicated button. The region is typically 0.1 to 0.4.

The user may also display Δ CV (═ Δ k) by clicking on a button dedicated to this purpose. These are calculated from the arrangement of the upper and lower compounds.

The application communicates the minimum Δ CV (═ Δ k) in the form of "Min Δ CV". From this data, a warning message is displayed indicating to the user the level of difficulty of separation. For example, the manual mode may include three difficulty levels:

easy, corresponding to a "min Δ CV" of greater than 4,

-a standard, corresponding to a "min Δ CV" of 1.5 to 4,

difficult, corresponding to a "min Δ CV" of less than 1.5.

(3) Amount of input solvent and sample to be purified:

the user is asked to enter the amount of sample to be purified and to select the solvents used (up to two) and their respective percentage ratios. For each solvent, the user can indicate if there are additions and add comments about them, or add comments about them on the underlying screen in a more general manner, if he so desires.

A screen summarizing his data is then displayed.

(4) This information is transmitted directly to the chromatography system safely:

the data may be stored in the user's personal database, sent to the user by email, or sent to the preparative liquid chromatography system via bluetooth or Wi-Fi.

(5) Pairing an application with a preparative liquid chromatography system:

the application includes a parameterization module that can pair the chromatography system with the application via File Transfer Protocol (FTP) via Wi-Fi, where an archive of analytical data is archived and read. This parameterization module may also enable the user to enter his email address to receive the file.

(6) Archiving and repeated sending of the data:

the application advantageously includes an archiving module in which the user can archive his data if desired. In this case, data will only be able to be queried from his smartphone. The user can send the analytical data of the TLC plate to the chromatography system again and as many times as he wishes, if necessary.

In an advantageous manner, the sending is done by means of a json file containing a photograph of the plate, the name that has been attributed to it, the Rf of the compound of interest, the general comments on the plate, an indication of the amount of product to be purified, the name of the solvent, their% proportions, the mention of additives and possible comments on the solvent (if applicable). All of these data were read by the processing algorithms described below to elaborate the recommendations for preparing liquid chromatography protocols.

However, unsatisfactory TLC separations may occur, for example due to the position of peaks of interest, due to the nature or amount of solvent to be purified being greater than the user's inventory, due to the fact that there are no suitable columns in inventory, etc.

In this case, the processor starts an algorithm which, by varying the analysis conditions (in particular the strong solvent or solvent pair used), makes it possible to obtain a separation different from the initial one at isocratic elution (block 5). This makes it possible to determine experimental conditions with the best potential in the optimal retention area to obtain efficient purification. For this purpose, the algorithm is based on the general diagram described in the principle of adsorption chromatography, r.l. snyder, ed.m. dekker (1968).

The algorithm calculates a limit value for the percentage of strong solvent specific to each of the five working amplitudes described above.

Preparative liquid chromatography conditions are then proposed (block 6 described earlier).

In the case where no TLC data is analyzed, but if an analytical chromatogram is available (block 9), another module is used, as described below.

In the case of chromatograms obtained by HPLC, the degree of separation between the two peaks is defined by the following relationship:

Rs＝2x(t_R2-t_R1)/(ω₂-ω₁)，

wherein t is_RIs the retention time of the peak and ω is the width of the bottom of the peak.

The conclusion is that the larger the Rs, the better the separation between the two peaks.

In the first case (block 10), the analytical stationary phase is the same as the analytical stationary phase of at least one column of the list of columns. From the input analytical columns, the algorithm identifies one or more columns in the list of columns that are maintained with respect to the material of the stationary phase, its trade name, and its grafted fields. The amount of sample to be purified is input.

Another algorithm (block 11) selects a column that is compatible with the system in terms of pressure, while taking into account the properties of the stationary phase, the size of the particles, the properties of the solvent, and the material of the column.

The algorithm calculates the magnification ratio based on the internal diameter and length of the column and the pore volume percentage of the stationary phase particles used.

The original sample mass to be purified is divided by the amplification ratio to calculate the number of injections to be performed.

Finally, the algorithm performs the transpose from the gradient method of the analytical column to the gradient method of the preparative liquid chromatography column. This transposition is based on a direct ratio of the column dimensions, recalculation of the gradient slope, its step size and its time, and the percentage of pore volume of the particles used. It also takes into account the hold up volume of the column V0 and the delay volume of the device. It therefore predicts the evolution of pressure and the consumption of solvent. Analytical retention times for compounds entered into the tables to calculate retention times were similarly converted to predicted retention times on preparative liquid chromatography columns.

In the case where the user does not have a column with the stationary phase identical to the stationary phase evaluated analytically (block 12), the algorithm identifies in the list of columns whether the field relative to the stationary phase properties corresponds to (a) untreated silica, (b) grafted silica other than C18, or (C) C18 grafted silica.

In cases (a) and (b) (silica or grafted silica other than C18), the algorithm identifies whether a field relative to the type of grafting in the list of stationary phases and columns can be associated; positively, the algorithm proposes a direct transfer.

In case (C) (C18 grafted silica, which may differ greatly in selectivity due to the grafting carried out), a table has been previously constructed, called "Atom table", which summarizes the experimental data of hundreds of columns based on C18 grafted silica, for different manufactures and manufacturers. The algorithm normalizes the experimental values according to the extrema. For each analytical column and each qualification criterion, the euclidean distance with respect to all other columns is calculated. After normalization, each distance of each criterion is added together to become the reference value for that bin relative to all other bins. One of the criteria is to define a uniform parameter with respect to hydrophobicity.

The algorithm searches the Atom table for the field of stationary phase, the same grafting conformation and type as the input analytical column (block 13).

If so, the algorithm calculates the proximity (Euclidean distance) of the selectivities for all stationary phases in the Atom table. It sorts the responses by ascending order and only retains candidates with a proximity value less than 0.17.

The algorithm compares its results with the Atom table and, based on the number of injections and their presence in the column list, only retains six candidates according to the standard, which translates into the fact that they are in the user's actual inventory.

The following steps are the steps described for block 11.

In case the user does not have any suitable column (box 14), he is informed of the fact that he has to buy a suitable column. The following steps are the steps described for block 11, depending on the column purchased.

If a directly positive solution is not found (block 15), positive screening is performed (block 16). Such a screen consists in studying the separation of different selectivities in the normal phase in analytical columns from HPLC columns or flash chromatography columns.

At the end of the screening, the algorithm selects the column with the best purification potential (block 17). The algorithm may optionally propose elution conditions, making it possible to obtain different separations.

The algorithm then relies on the separated analytical chromatograms of the selected column (box 18).

In the first case (block 19), the analytical stationary phase is the same as the stationary phase of at least one column in the list of columns. From the input analytical columns, the algorithm identifies one or more columns in the list of columns that are maintained with respect to the material of the stationary phase, its configuration, and the grafted fields. The amount of sample to be purified is input.

Another algorithm (block 20) selects a column that is compatible with the system in terms of pressure, taking into account the properties of the stationary phase, the size of the particles, the properties of the solvent, and the material of the column.

The algorithm calculates the magnification ratio based on the internal diameter and length of the column and the pore volume percentage of the stationary phase particles used.

The original sample mass to be purified is divided by the amplification ratio to calculate the number of injections to be performed.

Finally, the algorithm performs the transpose from the gradient method of the analytical column to the gradient method of the preparative liquid chromatography column. This transposition is based on a direct ratio of the column dimensions, recalculation of the gradient slope, its step size and its time, and the percentage of pore volume of the particles used. It also takes into account the hold up volume of the column V0. It therefore predicts the evolution of pressure and the consumption of solvent. Analytical retention times for compounds entered into the tables to calculate retention times were converted in the same manner to predicted retention times on preparative liquid chromatography columns.

In the case where the user does not have a column with the stationary phase identical to the stationary phase evaluated analytically (block 21), the algorithm identifies in the list of columns whether the field relative to the stationary phase properties corresponds to (a) untreated silica, (b) grafted silica outside of C18, or (C) C18 grafted silica (block 22).

In cases (a) and (b) (silica or grafted silica other than C18), the algorithm identifies whether a field relative to the type of grafting in the list of stationary phases and columns can be associated; positively, the algorithm proposes a direct transfer (block 20).

In case (C) (C18 grafted silica, which may differ greatly in function due to the grafting carried out), a table has been previously constructed, called "Atom table", which summarizes the experimental data of hundreds of columns based on C18 grafted silica, of different manufactures and manufacturers. The algorithm normalizes the experimental values according to the extrema. For each analytical column and each qualification criterion, the euclidean distance with respect to all other columns is calculated. After normalization, each distance of each criterion is added together to become the reference value for that bin relative to all other bins. One of the criteria is to define a uniform parameter with respect to hydrophobicity.

The algorithm searches the Atom table for the field of stationary phase, the same grafting conformation and type as the input analytical column (block 13).

If so, the algorithm calculates the proximity (Euclidean distance) of the selectivities for all stationary phases in the Atom table. It sorts the responses by ascending order and only retains candidates with a proximity value less than 0.17.

The algorithm compares its results to the Atom table and, based on the number of injections and their presence in the column list, only retains six candidates according to the criteria, at least three of which are in the user's actual inventory.

The following steps are the steps described for block 20.

In case the user does not have any suitable column (box 23), he is informed of the fact that he has to buy a suitable column. The following steps are the steps described for block 20, depending on the column purchased.

If no protocol is found after the normal phase screening, but a reverse phase protocol is present (block 24), then a second series of cases involving non-polar or medium polarity (NP/MP) samples is entered.

According to a first possibility, an isolated analytical chromatogram can be obtained (block 25).

The implementation of the method according to blocks 26-30 is similar to that described above with reference to blocks 10-14 and will therefore not be described again.

In case no reverse phase scheme is available (block 31), the system suggests to conduct a study of the separation of different selectivities in the reverse phase from HPLC or flash chromatography systems (block 32).

The algorithm may determine in two steps the column with the most interesting separation potential from the set of columns for which the thermodynamic parameters p and q are known (block 33). The first step is to determine the mobile phase conditions for which the retention factor of the last of the compounds eluted from the mixture to be purified is about 10 and to make it possible to recalculate the isocratic elution conditions so that each of the other columns achieves this same objective. The second step is to sample the sample into each column under isocratic elution and then select the most relevant column by comparing chromatograms.

According to a first possibility, an isolated analytical chromatogram can be obtained (block 34).

The implementation of the method according to blocks 35-39 is similar to that described above with reference to blocks 10-14 and 26-30 and will therefore not be described again.

In all cases, once the selected column has been placed in the appropriate position in the chromatography system, the processor initiates the performance of the chromatography according to the calculated mode of operation (block 8).

In a particularly advantageous manner, preparative liquid chromatography can be accelerated by the algorithm described below.

This accelerated possibility, on the one hand, enables the user to meet the need to terminate the purification more quickly in order to be able to handle another task that has become a priority and/or, on the other hand, reduces the analysis time while estimating that an increase in the flow rate does not significantly affect the degree of separation of the critical pairs.

To this end, the algorithm evaluates the maximum acceptable flow rate and pressure factor of the ongoing purification (taking into account, inter alia, the limiting pressure of the relevant chromatographic system, the limiting pressure of the column and the potential dry load pressure) as a function of the difficulty of separation Δ k of the TLC analytical data and the Rs of the HPLC analytical data (block 40). The algorithm automatically adjusts the gradient method to new conditions in real time. The user may choose to trigger acceleration at any time during purification. This operation is reversible and takes place in a completely safe manner without any loss of product.

Reference to the literature

US 7,686,959