HK1118752B - Elements for separating substances by distributing between a stationary and a mobile phase, and method for the production of a separating device - Google Patents

Description

Element for separating substances by partitioning between a stationary phase and a mobile phase and method for preparing a separation device

The present invention relates to an element for separating substances by partitioning between a stationary phase and a mobile phase, in particular for chromatographic analysis, and a method for preparing a separation device comprising a stationary phase, in particular a chromatography column.

Chromatography is a physicochemical method of performing separations in which the substance to be separated is distributed between two phases, one of which is a stationary phase and the other, mobile phase, moves in a defined direction.

The stationary phase is one of the two phases that make up the chromatographic system. A solid phase (sorbent), a liquid phase (solvent) or a gel may be used as stationary phase. In the case of a liquid phase, this is applied to a solid (carrier) which may also participate in the separation process. In the case of the binding phase, the layer with separating properties is chemically bound to the support or to the inner surface of the capillary.

Within the scope of the present invention, the stationary phase also includes a quasi-stationary phase in liquid-liquid chromatography.

In order to protect the separation column from contamination during chromatographic separation, sometimes a front column (Vors) is provided in front of the separation columnule). Generally, the pre-column is packed with the same stationary phase as the separation column, or with a material having a lower retention.

In the isocratic analysis, the composition of the mobile phase remains unchanged throughout the elution operation.

In contrast, in gradient elution, the composition of the mobile phase changes continuously or alternatively stepwise (step gradient).

In fact, substances with different polarities are usually separated by chromatography over a wide range of polarities by gradient elution, since in this way selective separation can be achieved. In this case, the more polar mobile phase is added continuously or stepwise to the initially normally non-polar mobile phase (normal phase chromatography) or the less polar mobile phase is added to the more polar phase (reverse phase chromatography).

In order to form the mobile phase by gradient, for gradient elution, it is necessary for the chromatograph to have at least two pumps for different solvents or at least one low-pressure mixer and control software. Gradient separations are generally less stable than constant solvent separations.

Another disadvantage of gradient elution is that: after elution with, for example, a solvent mixture of increasing polarity, the chromatography system must be returned to the initial thermodynamic equilibrium for the initial non-polar mobile phase used in the next elution, i.e., by extensive washing with the non-polar phase (in this example), before the column can be used for the next gradient elution. The "backwash" is usually carried out with 15 to 20 column volumes, resulting in an extended total cycle time and a large solvent consumption.

Therefore, the separation of a large number of samples to be examined and separated by gradient elution has the disadvantage that the total cycle time is prolonged, for example, in quality control, but for daily investigation of a large number of samples, the time per analysis for good separation should of course be as short as possible, in economic terms.

Another disadvantage of gradient elution is that the ever-changing composition of the mobile phase means that certain detection principles cannot be employed. For example, when the composition of the mobile phase changes, it is substantially impossible to employ a detector based on measuring the refractive index.

Even the application range of commonly used UV/VIS detectors in gradient elution is greatly limited, because many mixed solvents and additives, such as methanol, THF, TFA, formic acid, acetic acid, etc., have strong absorption in the UV range (< 230nm) themselves.

Even for mass spectrometers which have recently been used as universal detectors, side effects occur in gradient elution, for example, there is a problem of ionization in different chemical environments, making quantitative analysis much more difficult.

The separation of substances by gradient elution also has ecological disadvantages: the mobile phase contained in the mixture after elution cannot be recovered or can only be recovered with difficulty, which requires costly disposal of the solvent mixture and the additional costs required for supplying (fresh) solvent to form the gradient. Moreover, gradient elution requires the use of much more costly, higher purity solvents than the constant solvent technique.

In addition, backwashing of the separation column also requires a considerable amount of solvent, which cannot be recovered.

In practice, and also in the context of preliminary experiments, the user first develops a column with the most suitable stationary phase from a column with a different stationary phase (which column is supplied in the form of a test column), and in practice the optimization of the separation technique is finally based on the optimization of the mobile phase, i.e. by selecting a suitable solvent, the pH of the mobile phase, other additives, etc., which in turn leads to an improved separation.

However, by empirically selecting the correct stationary phase, a defined selectivity can be achieved more simply. However, it is often the case that as soon as a separation is achieved between a particular pair of key peaks, another pair of peaks is incorporated into one peak elsewhere in the chromatogram.

To prevent this, it is proposed to use a mixture of suitable stationary phases (mixed bed) as the stationary phase; for certain separation problems in constant solvent analysis, selective separation is generally achieved using the stationary phase.

G.j.eppert and p.heitmann in lc.gc Europe, p.2, month 10 2003 mention optimization of mixed bed columns based on the following steps: the retention time of a test substance mixture is first determined using three or four test columns with different selectivities, and then the appropriate mixed bed column is calculated for the particular separation problem.

However, a disadvantage of these stationary phases in the form of mixed beds is that the column manufacturer has to accept a commission to pack (packer) the respective mixed bed column for each new separation problem. Thus, in the short term, the user is likely to be unable to perform a constant solvent assay with the desired selectivity. The column cannot be filled by the user, since the user does not have the necessary special equipment and lacks the relevant technical knowledge. In addition, it is also quite difficult to pack the mixed bed into the separation column with good reproducibility.

In multidimensional chromatography (multidimensional chromatography), it is known to provide column branches from a main column that are to be connected at different times, so that the individual fractions are then "split" out of the main column at defined points in time by the temporarily connected column. Finally, the separation of the mixture in multidimensional chromatography takes place only to a certain extent on the stationary phase of the separation device, while the main separation is effected by switching to a further separation device which is additionally supplied with eluent.

Even if good separation can be achieved using multi-dimensional chromatographic analysis, it is very expensive, prone to failure and only suitable for routine measurements at considerable cost.

To solve specific separation problems, such as the separation of 20 component amino acid mixtures, j.l. glajch et al in "Journal of Chromatography", 318(1985), p.23, propose to improve selectivity by combining a stationary phase and four mobile phases. However, this setup is based on purely empirical tests and has not been specifically calculated.

In order to achieve optimization of mobile phases in liquid chromatography, sz. nyiredy, b.meier, c.a.j. Erdelmeier, o.sticker proposed the so-called "prism" mode in "High Resolution chromatography" & chromatography. communications "8, (1985). In this mode, the solvent intensity is plotted vertically, while the ratio of the components is plotted in two dimensions in the horizontal plane, which mainly affects the selectivity.

Subsequently, in the column liquid chromatography analysis, the global optimization of the mobile phase was predicted from 15 individual measurements, and the local optimization of the mobile phase was predicted from 12 individual measurements (sz. nyiredy, w. wosniok, h. thiele, o. study, "Fluid chromatography.

This model is also used to achieve optimisation of the extractant in liquid-liquid extraction, in which case the optimum extractant can be determined from 12 measurements (sz. nyiredy, "Chromatographia", 51, 288 (2000)).

The problem underlying the present invention is to provide an apparatus and a method for solving the problem of chromatographic separations that are fast, efficient, environmentally friendly and have optimal selectivity and detection capabilities.

This problem is solved by the features of claims 1 and 15.

According to the invention, a Set (Set) of different separation elements is provided, whereby separation devices with different stationary phases can be constructed, said construction being performed by joining or connecting together the different separation elements.

The kit of the invention comprises a plurality of different stationary phases S which can be linked or connected together_i(i.gtoreq.2) separation elements, at least three of which contain different stationary phases S_i。

The coupling or connection is performed by coupling or connecting together such separation elements, in particular carrier elements which together with the respective stationary phase form the separation element. For example, by segmenting the column (S)ulensegment) are joined together at their respective tube ends to join the column sections.

It is also possible to couple the separate elements by means of another coupling or connector.

Within the scope of the invention if two stationary phases S_AAnd S_BThe two stationary phases differ in selectivity.

The smallest kit therefore comprises in each case three separate elements or column sections S_AAnd in each case three separating elements or column sections S_BWherein A and B are stationary phases having different selectivities.

Preferably, the kit comprises at least three different stationary phases S_A、S_BAnd S_COf the separating element or column section, sleeveIt is preferred to include 3 of each of these separating elements, preferably at least 5, preferably at least 7, particularly preferably in each case 10 or more.

The individual stationary phases should be selected so that they have different physicochemical properties.

Optionally, in addition to or instead of the three different types of separation elements described above, the kit may comprise separation elements having, for example, a stationary phase comprising a saturated or unsaturated alkyl group (C1, C2, C3, C4, C6, C8, C12, C14, C16, C18, C22, C27, C30), which may be monocyclic, polycyclic or acyclic, alicyclic or heterocyclic; nitro, cyano, carbonyl, carboxyl, hydroxyl, diol or thiol groups, glycidoxy, optionally etherified; amino or chiral groups, amide groups, carbamates, ureas, perfluoroalkyl or perfluoroaryl groups and other haloalkyl and haloaryl groups, polybutadiene groups or other organic polymers, affinity chromatography modifications (affnit)tschromoprophish modifiering) or ion-exchangeable groups (cation exchangers, anion exchangers, zwitterions), unmodified support materials (e.g. silica gels), molecularly imprinted polymers (moleklar gelr)gte polymer, MIP), or combinations thereof, such as a multifunctional stationary phase.

Unlike multidimensional chromatography, the substances are separated on an optimized basis on a coupled or connected separation device or column, i.e., the components of the mixture pass through the entire separation device. The separation elements that are bonded together remain interconnected at least throughout the separation-bonding or debonding (Abkopplung) during the separation process, which is well known in multidimensional chromatography, does not occur.

Another important point is that the ratio of the stationary phases of the coupled separation devices to each other is variable, since the separation devices can again be disassembled into individual separation elements, and separation devices with different ratios of the stationary phases to each other and different selectivities can be constructed from these individual separation elements.

Unlike known mixed bed columns, the columns required for the present invention are constructed by mechanically linking individual ready-to-use separation elements, i.e., typically by joining or linking together carrier elements (e.g., segments of a tube as column segments). This does not change the stationary phase, they remain unchanged in the separation element, in particular they do not mix together.

Unlike the preparation of pre-calculated mixed bed columns, according to the invention the user can prepare the desired separation equipment in situ by simply mechanically coupling the desired column sections, whereas the former ultimately makes it necessary for the column manufacturer to pack the column according to the specific user requirements, i.e. to prepare and mix the specific stationary phase.

The invention also relates to a method for producing an optimized device for separating substance mixtures.

Firstly, for the at least two stationary phases S_i(i.gtoreq.2) for each of the components, the relative retention factor k for each component is determined by the following formula

k＝(t_R-t_M)/(t_M-t_M ^Ec) (1)

(t_R: retention time, t_m: flow time, t_m ^Ec: additional column volume correction). The additional column volume correction takes into account the feed line volume, injector volume and detector cell volume contributions to time. It is recommended to determine the mobile phase such that the K value lies between the earliest and the last eluted substances, ranging from 1 to 20.

The base measurement should preferably be on each separation element (e.g., S)_A、S_B、S_C) Or on several similar or different coupling elements (e.g. 10 coupling elements S)_A(ii) a Or 5S_A3, S_B2, S_C(ii) a 3S_A、5S_B、2S_C(ii) a And 2S_A、3S_BAnd 5S_B) The process is carried out. The base measurement may also be performed on a separate base measurement column.

Subsequently, according to the ratio of each component to be separated and having a stationary phase S_iThen calculating an optimal combination of separation elements and their sizes for a particular separation problem, and then joining or otherwise connecting the separation elements having the predetermined sizes in the manner previously calculated into the optimized separation apparatus.

The size of the separation element means the volume of the respective stationary phase in the separation element. When the separating element has a constant diameter, the dimensions and length are proportional. If all the separating elements have the same diameter and the same length, the size is proportional to the number N of separating elements to be joined.

The most suitable combination of separating elements for a particular separation problem is preferably calculated using a prism model. Preferably, the retention factor k determined from said base measurements is based on the stationary phase and its size for any feasible combination_iA predetermined number N of separating elements to be connected, in particular a predetermined total number N of separating elements of X + Y + Z, is defined, and the respective retention factors of the components to be separated at the selectivity point XYZ, i.e. the stationary phase S, are calculated therefrom_iRetention factor k of each of the heterogeneous combinations of_xyz：

k_xyz＝(X×k_A+Y×k_B+Z×k_C)/(X+Y+Z) (2)

Wherein X represents a separating element S_AY denotes a separating member S_BZ denotes a separating element S_COf the respective number of the first and second.

From the plurality of calculated coupling possibilities XYZ, the optimum selectivity alpha or the optimum resolution R of the separation element or segment is determined_sThen, the individual elements or column sections are joined together to form a separation device, in particular a column, optimized for the separation problem and comprising N separation elements, wherein α is determined by the following formula:

α＝k₂/k₁ (3)

R_sis determined by the following formula:

R_S＝2*(t_R2-t_R1)/(W_1/2 1+W_1/2 2) (4)

wherein t is_R1Is the retention time, W, of the respective compound_1/21 is the half peak of each compound.

By using a specific calculated coupling of the separation elements, in particular the column sections, the stationary phase can be optimized for a specific separation problem, so that elution with a continuously or stepwise changing mobile phase is no longer necessary, or the manufacturer is no longer required to pack a specific mixed bed column.

If the underlying measurements have shown that the required separation can be achieved by only one type of column section, the remainder of the calculation can be used to optimize the length of the column and to construct the column from only that one type of column section.

Otherwise, the various possible column section combinations for a given number N of column sections are calculated until the desired separation is obtained. When the desired selectivity a for one or more pairs of key peaks is reached_critOr resolution R_s，critThe optimization and calculation may be terminated.

If, within the range of the results of the calculations for each possible combination of, for example, three types of column sections and a total number of column sections N, a sufficiently good separation is not calculated, i.e. no specific target is obtainedR below the value defined by the separation problem_s，critThen the number N of column sections is changed (in particular increased) and the changed number N' is calculated again.

If, within the range of calculations performed on the N column sections, no satisfactory separation is predicted, then a new calculation is performed, adding one or more additional stationary phases.

For this purpose, the additional column section S is used_jAdditionally, a base measurement is made and then a new calculation is made.

If the separation capacity is insufficient for a given combination, the resolution can be increased by increasing the total number of column segments N by the existing ratio. In fact, if the separation is too good, the number of segments can be reduced by the same ratio in order to shorten the total cycle time.

The separation apparatus optimized for this particular separation problem is then joined or connected by a calculated number and/or size of separation elements and a corresponding separation is carried out.

Based on the calculations, the elution order and the resolution of the peaks can be predicted.

The separation device, in particular the column, which is optimized for this particular separation problem, can thus be assembled quickly and efficiently from the set of separation elements, usually on the basis of only three (|) basic measurements.

Although usually three different stationary phases are sufficient to achieve a sufficiently efficient, in particular constant solvent separation, the number of stationary phases can of course be further increased to four, five or more, the formula for calculating the heterophase retention factor K in the numerator and denominator extending correspondingly to n stationary phases.

If the stationary phase or the separation element comprising the stationary phase does not have the same dimensions (e.g., diameter and length of the packed column section), the retention factor for each component can be calculated from the following equation:

K_αβγ＝(α×k_A+β×k_B+γ×k_C)/(α+β+γ) (5)

where α, β, γ correspond to the total internal volume of the separation element filled with stationary phases A, B and C in the heterogeneous phase combination. K_A，B，CAs already described-is the retention factor determined from the individual base measurements.

If a guard column has to be provided before the separation device in the subsequent measurement, this has to be taken into account in the basic measurement and calculation.

Another advantage is that the first column section can also be considered and used as a protective column and can be replaced very easily if desired. Accordingly, additional protection columns need to be employed.

In addition, the method according to the invention can also be carried out in variant form, so that-without specifying specific dimensions of the separation element-the optimized separation apparatus is calculated from the basic measurement results, and the optimized column can then be filled accordingly by the manufacturer on the basis of the previously calculated proportions of the individual stationary phases. In this case, the calculation provides the volume of each stationary phase optimized for selectivity, and the respective volumes determined are subsequently packed into a suitable carrier element, for example a suitable column.

In general, when using the device according to the invention and the method according to the invention, it is not the mobile phase which undergoes a continuously changing composition, but the separation device (in particular the column) specifically assembled for the separation by coupling the individual separation elements has a changing composition necessary for the selective separation, which is based on the separation elements or segments having different selectivities, that is to say the separation device has a stationary phase gradient.

Accordingly, the separation carried out with the apparatus of the invention can also be carried out in the form of a constant solvent and with the desired selectivity, thus avoiding the disadvantages of gradient elution. Thus, no backwash is required, i.e., solvent is saved and overall cycle time is shortened. In addition, the stationary phase or at least the major fraction not contaminated with substances of different polarity can be recycled in the isocratic elution, which additionally saves disposal costs and the costs of providing fresh solvent and is therefore also beneficial to the environment.

For this purpose, the individual regions of the mobile phase which are loaded with the individual separated substances are preferably removed, while the remaining solvent is captured, recycled and reused. Especially in the field of preparative chromatography using relatively large amounts of solvent, the reusability of solvent by means of solvent-invariant chromatography not only provides a significant environmental benefit, but also a considerable cost saving, since the costs for disposal and supply of fresh solvent are significantly reduced.

Another important advantage of the present invention is that when using a constant solvent elution, more different detection principles and detectors can be used to determine the various substances.

Since the composition of the mobile phase remains constant during constant solvent elution, the differential refractometer can be reused for universal detection of substances.

Typically, the solvothermal elution and separation of substances using the kits of the invention can be carried out in acetonitrile/water mixtures, for which case the increase in absorption in the UV range is not initiated until 190nm wavelength (50% transmission). Accordingly, due to the constant solvent elution, substances with chromophores can be detected even in the short-wave UV region.

Thus, the separation according to the invention can be performed in the form of a constant solvent, so that other, generally more suitable, more sensitive and/or less expensive detection methods can be employed.

Since the composition of the mobile phase remains constant over time, and moreover, the baseline is of course more stable in each detection method, the effects due to mixing are also avoided.

Constant solvent separation also has a good effect on the mass spectral quality because the ionization remains constant and, in addition, no gradient-induced side effects (steady and constant background) occur.

Furthermore, for detection in a constant solvent elution, an electrochemical detector or a conductivity detector may also be used.

Since the use of the chemostat assay makes almost all detection principles now available, it becomes possible to couple the various detectors in series, thereby improving the characterization of the substance.

In addition, with the apparatus of the present invention, the failure-prone devices required to provide the gradient, such as additional pumps and gradient mixers, are not required, thereby reducing not only the acquisition costs of the chromatograph, but also the operating costs.

In the fields of nanoscale, microscale, analytical and preparative applications, and in the field of sample preparation and/or purification by solid-phase extraction or filtration, the separation element kit of the invention and the method of the invention can be used in each case for all of the following methods: the methods employ physicochemical separations based on different interactions of at least two phases, such as Gas Chromatography (GC), Liquid Chromatography (LC) (column or planar), chromatography using supercritical gases as mobile phases (supercritical fluid chromatography, SFC), electrophoretic separation techniques.

In liquid chromatography, the following methods are also employed: the methods are performed under reduced pressure (e.g., flash chromatography) or at elevated pressure (e.g., HPLC, MPLC), or separation using different interaction mechanisms, such as reverse phase liquid chromatography (RP), normal phase chromatography (NP), Ion Chromatography (IC), size exclusion chromatography (SEC, GPC), or hydrophobic interaction chromatography (HIC, HILIC).

The separation element of the stationary phase may be, for example, a section of a column, a TLC plate or a section of an electrophoresis gel. Although the invention has been described primarily with reference to columns and column sections, the references are equally applicable to other separation elements or segmented separation devices or segmentable separation devices, such as TLC plates, which may be adapted to any possible length of the stationary phase of interest.

The association of the stationary phase is also independent of the type of support material used (silica gel, polymers, metal oxides, glass, quartz glass, polysaccharides, agar gel, carbon, etc.) and can also be applied to chromatographic methods of various orders of magnitude, ranging from micro-nanoscale methods to preparative methods.

In addition, the stationary phase may be either particulate, fibrous or fibriform, or may be monolithic. The advantage of using monolithic stationary phases is that they are very easy to bind, since the stationary phases do not need to be retained by frits or screens.

The invention is also particularly advantageous when used in nanotechnology, since it is extremely difficult to reproducibly provide a gradient in the mobile phase in such small quantities.

Of course, the kit of the present invention may also be adapted to a particular separation technique, or may contain a particular segment for a particular separation problem, as an additional segment if necessary.

Thus, in ion chromatography or ion pair chromatography, it is also possible to construct the stationary phase from a column section, or for example, a column section for ion pair chromatography and other column sections (e.g., polar column sections) may also be joined together.

Another advantage of using a separate device that is specifically pre-calculated and then coupled or assembled is that the overall cycle time is reduced. The use of separate elements and the calculation of the individual separate elements to be joined together and the number thereof also finally makes it possible to optimize the total cycle time, i.e. as short as possible, but long enough to achieve the desired separation.

The kit may also include a section for the directed adsorption of certain substances, as in solid phase extraction. From the mixture to be separated, the target substance is almost quantitatively adsorbed in this specific adsorption section and is thus also enriched in this section, and is subsequently desorbed again. Instead of only one segment, as is customary until now, it is possible to foresee the realization of a true cascade of segments in order to achieve a targeted sequential adsorption of different substances.

The present invention can also be used in other analytical techniques, such as in electrochromatography, which separates mixtures of substances based on electroosmotic flow, or electrophoresis.

The kits of the invention and the methods of the invention are suitable for on-line continuously operated separation processes and/or methods with mobile phase and/or constant rate, such as HPLC, on-line TLC and on-line OPLC.

The kit of the invention may also comprise at least two different TLC plates, each plate then being included in the kit in a larger number, in particular at least 3 plates each. The TLC plate may be adapted to an optimal length and the various TLC plate sections may then be joined together to form a plate formed from the various sections and finally form a plate with a gradient in the stationary phase.

In particular, the kit allows the user to build in situ a separation device particularly suitable for their (routine) analysis, and then to perform the separation with the advantages of constant solvent elution already described.

The advantages of an optimized separation device are particularly realized in the case of an optimized separation device for a continuous separation process (i.e. a constant solvent separation process) and having a stationary phase throughout the separation process (i.e. not even "switching" the column).

In particular applications, besides optimization of the stationary phase, it is of course also possible to optimize the mobile phase for the targeted separation, for example with gradients.

The specific design of a specific column section will of course depend on the kind of separation technique and the amount of substances that will be separated using the separation device prepared from the separation elements that will be joined together.

The length of each individual separation element depends on the subsequent total length of the device (especially the column) containing the stationary phase, the number of stages required and the particular separation problem. In case the separation element is a segment of an analytical LC column, the length of the segment is at least 0.5cm, preferably at least 1.0cm, to simply mechanically join the segments together. The length of the column section is preferably 2cm, so that a column of 20cm in length can be constructed from 10 joined sections. Particularly preferably, the length of the column section is 1cm or integral multiple thereof, and can be up to 500 cm; preferably, the length of the column section is no greater than 12.5 cm. In capillary gas chromatography (cGC), column lengths of up to 100m are contemplated, and preferably 25 m.

The column section may be straight cylindrical or spiral cylindrical (especially in the case of a long column section, e.g. for GC), or may be of some other shape.

The internal diameter of the column may vary considerably depending on the amount of substance to be separated and the particular chromatographic process, and may be of the order of 20 microns to 2 meters, typically 20 microns to 10 millimeters in the case of analytical columns. For separation purposes, columns with an internal diameter of 2mm to 1m are used.

In principle, the kit may also additionally comprise a "multiplier section". These "multiple segments" are column segments that define the stationary phase, and also exist as "single segments," but the length of the "multiple segments" is an integer multiple of the length of a single segment. For example, in addition to 10 segments having a length of 2cm and having a phase comprising phenyl groups, the kit may also include a "double segment" having a length of 4cm and having a phase comprising phenyl groups, and a triple segment having a length of 6cm and having a phase comprising phenyl groups; when these multiple sections are additionally present, the time required to join and disconnect the sections can be reduced.

By theoretical considerations based on prism models it was found that when in each case in addition to a column segment of length 1, a column segment of each type of stationary phase with the smallest possible length (e.g. 1/2 or 1/4) is included in the calculation, a much wider selective optimization of the working range can be covered by a reasonable number of calculations than if, for example, the total number N of column segments is increased overall by one or two segments.

The separating elements should be coupled as far as possible by means of inert, dead-space-free connections (veribingdung), for example by:

capillary joints

Directly by means of couplings with small dead spaces

In the case of a capillary column, the ends of the capillary are butted directly together by joining them together using a coupling

In the case of capillary columns as well, by shrink fitting the ends of the capillary via a shrinkable sleeve (Schrumpfschlauch)

Screw-type joining of the column ends together using a washer with a through-hole

By a screw connection with capillary holes

Directly bringing together the filling end of one separating element and the filling end of the other separating element

By packing the stationary phase in the column tube in stages

Preferably, each separating element has the same coupling means (Kopplungsmittel) at each end, so that any end of one column section can be connected to any end of another column section.

Another preferred embodiment contemplates providing mutually different coupling means (male, female) at each end of the column section. In this case, the end of one column section is inserted at the beginning of the next column section, so that the inner diameter of the filling cross section and thus the linear flow velocity is kept constant. In this way, no turbulence is formed at the junction. Other advantages of such a system are the compartment-type sealing (gekammerte Dichtung) at the junction point and the protected filling of the individual separating elements. In addition, the filling is internally closed, protected. Simple manipulations by mutual insertion are particularly advantageous. The most important point of this coupling is, however, that the coupling is substantially free of dead spaces, since the ends of the separating element, with the thin frit, are pressed directly onto the screen with the desired cross-flow filtration (Kreuzfiltration).

It is also possible to provide a one-piece seal at each end of the column section, preferably 0.5-1mm thick. These column sections are joined in a similar manner (convex, concave) as described above. In this case, there is an advantage in that any metallic terminals such as a sieve and a frit are not required. In addition, the monolithic seal can be chemically modified and thus can be compatible with the particular stationary phase used. With such a system, two separate elements can be joined together without the use of transition elements (e.g., screens and frits).

If chromatography plates shortened to a suitable length (size) are used as separation elements, for example in TLC, OPLC or HPTLC, the separation elements can be joined by overlapping the stationary phases of two plates at the plate ends, so that the stationary phase of a first plate faces, for example, upwards, the stationary phase of the next plate faces downwards, etc., or the plates are placed adjacently and then joined via an inert bridge which then covers the stationary phases of the two plates.

The process of the invention will now be explained in more detail with reference to application examples.

I. Based on the prism model, an embodiment of an optimized combination of target separation elements (cylinder segments) is calculated:

a) for methyl paraben, acetophenone, ethyl paraben, dimethyl phthalate A mixture of ester, 2, 3-dimethylphenol, methyl benzoate and anisole, when used as 10 of the acetonitrile/water 30: 70(v/v) of the solvent have cyano and/or phenyl and/or C18 groups The best possible separation when combining column segments of clusters.

In each case, the kit comprises at least 10 CN asIs a stationary phase (S)_CN) The column section of (2) and 10 phenyl groups as stationary phase S_PHAnd 10C 18 as stationary phase (S)_C18) The column section of (1).

For each of these three different stationary phases, a respective retention factor k was determined according to (1) in the range of the base measurement for each of the 7 different substances to be separated, as shown in the following table:

	components	KCN	KPH	KC18
					1	Hydroxybenzoic acid methyl ester	2.4	5.0	10.2
2	Acetophenone	2.8	7.0	8.6
					3	Hydroxybenzoic acid ethyl ester	3.1	7.7	20.1
4	Phthalic acid dimethyl ester	3.2	9.6	10.5
					5	2, 3-dimethylphenol	3.5	9.7	26.3
6	Benzoic acid methyl ester	3.5	11.0	17.2
					7	Phenylmethyl ether	3.7	11.8	19.0

In the present embodiment, the total number N of column sections to be joined together is set to 10. For each hetero-phase association of the column sections, i.e. X stationary phases S_CNY stationary phases S_PHAnd Z stationary phases S_C18And wherein for N-10-X + Y + Z, the retention factor of the heteroleptic junction at the selective point XYZ is calculated according to (2):

k_xyz＝(X×k_CN+Y×k_PH+Z×k_C18)/(X+Y+Z) (6)

the selectivity of all adjacent pairs of peaks is calculated from the retention factor calculation of the individual junctions XYZ of the separation elements. The optimal selectivity point is the XYZ point where all pairs of peaks achieve baseline separation. If multiple selectivity points achieve baseline separation, then the ideal point is the point that would otherwise have the smallest retention factor for the last eluted component.

The value used for baseline separation, i.e., the point at which the peak reaches the baseline again, depends on the particular chromatographic technique or separation efficiency that can be achieved by the particular separation method. In the case of HPLC, to meet the requirement of good resolution, the value should be about 1.5, about 1.05 for GC and greater than 1.3 for TLC.

The selectivity points 442 are illustrated from the following (i.e., 4 segments S)_CN4 segments S_PHAnd 2 segments S_C18) The comparison of the calculated retention factor and the measured retention factor shows that the average deviation of the calculated retention factor and the measured retention factor is about 4% on average:

	1	2	3	4	5	6	7
								Kmeasuring	6.59	7.01	9.04	11.77	12.22	13.29	15.11
KComputing	6.31	6.86	8.80	11.07	11.81	12.82	14.21
								Deviation [% ]]	4.5	2.2	2.7	6.3	3.5	3.6	6.3

The separation selectivity depends on how many separation elements or column sections with different stationary phases (or, for the latter, with different polarities) can be joined together, the total number N of column sections to be joined, and others.

a2) For the mixture of 8 triazines, it was determined that when methanol/water 50: 50(v/v) was used Is a solvent, has 10 cyano and/or phenyl groups each and/or C18 groups each and/or has polar groups The best possible separation is achieved when the column segments of the C18 group of the amide group are combined.

In each case, the kit comprises at least 10 CNs as stationary phase (S)_CN) The column section of (2) and 10 phenyl groups as stationary phase S_PHAs the stationary phase (S) with 10C 18_C18) And 10 of the column segments having a polar insertion C18 as the stationary phase (S)_peC18) The column section of (1).

For each of these 4 different stationary phases, a respective retention factor k was determined according to (1) for each of the 8 different substances to be separated within the range of the basic measurement, as shown in the following table:

	components	kCN	kPH	kC18	kpeC18
						1	Prometone	1.16	4.57	6.09	4.51
2	Simetryn	1.28	6.82	11.85	7.55

3

Ametryn

1.41

7.73

12.18

7.42

4	Prometryn	1.45	10.41	22.82	14.19
						5	Terbutryn	1.39	10.15	19.70	11.16
6	Simazine	1.61	11.85	24.03	15.72
						7	Atrazine	1.78	18.57	46.50	27.22
8	Propazine	1.97	21.69	55.86	27.85

The chromatogram of these values is shown in FIG. 1. It has been found that the single separation column by itself does not solve the separation problem.

FIG. 1 separation of triazines in different stationary phases

The optimum composition of the stationary phase can be determined from the calculation of the k values and selectivities for all possible combinations. In this case, it comprises a 180mm phenyl phase, a 60mm C18 phase with polar insertion groups, and a 60mm cyano phase.

The retention factor calculations and measurements for the optimum column composition are compared in the table below. The deviation was 3.4% on average.

	1	2	3	4	5	6	7	8
									KMeasuring	4.30	6.42	6.90	9.09	9.96	10.75	17.21	20.19
KComputing	4.30	6 47	7.06	9.45	10.29	11.60	18.52	20.74

Deviation [% ]]

0.0

0.8

2.3

3.8

3.2

7.3

7.1

2.7

The calculated and measured chromatograms are compared in fig. 2. It can clearly be seen that they fit very well.

Figure 2, isolation of triazine: measured and predicted

b) Number of possible combinations

I) From S to 3 different column segments of the same size (S)_A、S_BAnd S_C(ii) a Herein is S_CN、S_PHAnd S_C18) And a total of N-10 linked column segments, if only two of the three different column segments are linked together, i.e. only S_A、S_BOr S_A、S_COr S_B、S_CThen 27 different combinations were obtained. If all three different column sections S_A、S_B、S_CAre connected togetherThen there are another 36 possible combinations.

Thus, in general, there are 63 possible combinations for the case of column sections of three different polarities and a total number of column sections to be joined of 10.

II) if the columns to be combined are not exactly 10, but the total number N is in the range of 6-15, i.e. N-6-15 and s-3, the number of possible combinations is increased to 625.

III) calculation of the above possible combinations (I), i.e. N ═ 10 and for the three stationary phases, when the stationary phase S is added_DI.e. s-4, feasible combinations with 2, 3 or 4 stationary phases are 960.

IV) if based on III), the total number extends to at least 6 to at most 15, and then a total of 2500 possibilities are possible when s is 4 and in each case a 3-phase combination.

The possible combinations are further increased by extending the number of available stationary phases combined with each other, for example, to 5 or more (e.g., combinations in which no more than three or four different stationary phases are associated). Additional "fine tuning" can be obtained by considering either 1/2 or 1/4 column segments in the calculations.

The invention is described below on the basis of a preferred application example of a coupling column section.

The column comprises one or more column sections, and a clamp, which in turn comprises individual clamping elements that can be screwed together and, if desired, can be screwed onto connectors at the top and bottom of the individual columns.

Independent of the specific preferred embodiment described in detail below, the individual clamping elements can also be connected by means of plug connectors or other types of connectors. Furthermore, it is also possible to dispense with the individual clamping elements altogether, but to insert the column segments into a guide sleeve of suitable length with a threaded end, and then to press the individual column segments together axially with a connector that can be screwed onto said guide sleeve.

The column sections may also be pressed together axially by hydraulic means in a suitable device.

The following particularly preferred embodiments are important: even at the junction, the inner diameter of the column remains constant, and thus the flow rate is constant throughout the column.

In addition, the post coupling described below may also be generally used to couple posts, such as front posts and main posts.

The figures show:

FIG. 1: a) an exploded view of the column section, and b) a schematic view of the complete column section,

FIG. 2: fig. 1b) in the clamping element 30 (step 1), in the further clamping element 30 '(step 2), in the further column section 11' (step 3), in the assembly of the column section

FIG. 3: a schematic representation of the top column section 11 or top clamping element 30 and the bottom column section 11 "or bottom clamping element 30" being coupled to respective adapters, an

FIG. 4: the column segments and adapters are joined together to form a schematic representation of the column.

The column section 11 (see fig. 1) comprises a column tube 13 with a stationary phase 12. The vial 13 may be made of, for example, metal, plastic, ceramic, or a combination thereof, having a predetermined outer profile (with steps 22, 21) and a constant inner diameter d_i。

At the top end 14, the tube 13 has a defined groove 15, the diameter d of which₁₅Must be larger than the inner diameter d_i. The groove 15 has a shoulder for mounting a seal 17. The seal 17 may be made of, for example, plastic, metal, or some other material suitable for sealing. The recess 15 accommodates a filter or filter-screen combination 16, said combination 16 protecting the stationary phase and having an annular seal 17 thereon. The wall 18 of the recess 15 is in contact with the other columnThe segments 11' simultaneously serve as guide rails during the joining process or when assembling the male adapter 40 (described below).

The tube 13 is shaped at its bottom end 19 by a male connector 21 and closed by a porous frit 20, the purpose of said porous frit 20 being to clamp the stationary phase 12 in the tube 13. The porous frit 20 may be a metal, plastic, ceramic, sintered silica gel, monolithic silica gel, or monolithic polymer, optionally surface modified.

A clamping member 30 (fig. 2) receives the post segment 11 and its coupling. The clamping element 30 may be metal, plastic, ceramic, or a combination thereof. At its top end 31, it has an internal thread 32 and at its bottom end 33 an external thread 34. Inside the clamping element 30, there is a through-hole 35 with a step 36, in which the column section 11 fits. The step 22 of the column section 11 rests on the step 36 of the clamping element 30.

The outer surface 37 of the gripping element 30 may be embossed diagonally or axially to facilitate handling, or may have a surface for use with a wrench.

Fig. 2 and 3 illustrate the assembly of column sections:

1. the column section 11 is placed in the clamping element 30 in the manner described above (arrow 1 in fig. 2).

2. The other clamping element 30' is screwed completely into the first clamping element 30 at the top end 31 of the first clamping element 30 (arrow 2 in fig. 2).

3. The other column section 11 'is placed in the threaded clamping element 30' (arrow 3 in fig. 2).

4. Steps 2 and 3 are repeated until the desired number of column sections 30 is reached.

5. The male adaptor 40 is inserted into the recess 15 of the top column section 11 in fig. 3, and the adaptor screw 41 is screwed into the clamping element 30.

6. At the bottom end 19 of the bottom column section 11 ", a female adapter 42 is fitted over the protrusion 21" and screwed onto the clamping element 30 with an adapter nut 43.

7. The resulting segmented column can now be connected to a chromatography system via capillary connections (stainless steel, plastic or sintered silica).

In the preferred embodiment described above, if, for example, a double length column section is to be connected to other column sections, the double length column section can be received in two clamping elements that are connected to each other and then joined.

Claims (13)

1. Kit of parts for separating substances by partitioning between a stationary phase and a mobile phase,

the kit comprising at least six separate elements to be joined or connected together to form a separation apparatus,

said separation element is called T_iWhere i.gtoreq.2, the separating element T_iEach comprising any stationary phase and a support member, and

said separating element T_iComprising at least two different stationary phases S_iWherein i is more than or equal to 2;

and having a defined stationary phase S_iEach separation element T of_iIn other words, the kit comprises at least three separating elements T_i。

2. The kit of claim 1, characterized in that the coupling or connecting together of the separate elements is achieved by coupling or connecting carrier elements.

3. Kit according to claim 1 or 2, characterized in that the kit comprises a stationary phase, S, having at least three selectively different selectivities_A、S_BAnd S_CThe separation element of (1), for each of the three separation elements, comprising at least 5 in the kit.

4. Kit according to claim 1 or 2, characterized in that the stationary phase of the kit contains

Optionally etherified saturated or unsaturated alkyl, said alkyl being optionally monocyclic, polycyclic or acyclic, alicyclic or heterocyclic;

nitro, nitro,

A cyano group,

A carbonyl group,

A carboxyl group,

Optionally etherified hydroxy,

Optionally etherified diol, or thiol groups,

Epoxy propoxy;

amino or chiral group,

Amide group, amide group,

A carbamate,

Urea, urea,

Perfluoroalkyl or perfluoroaryl or other haloalkyl or haloaryl;

polybutadiene groups or other organic polymers,

Affinity chromatography modifications or ion exchangeable groups,

An unmodified support material or a molecularly imprinted polymer, or a combination thereof.

5. A kit according to claim 1 or 2, characterized in that the separating element has a length of 0.5cm to 50 m.

6. A set according to claim 1 or 2, characterized in that the separating elements are column sections joined in a manner substantially free of any dead space.

7. A kit as claimed in claim 6, characterized in that the column sections comprise coupling means (15, 21) provided at the top and bottom of the column sections and differing from each other so that the bottom end of one column section can be inserted into the top end of another column section.

8. The kit according to claim 6, characterized in that the coupling of the column section (11) is performed by means of a clamping element (20), said column section accommodating said clamping element.

9. Use of a kit according to any of claims 1 to 8 in liquid chromatography, gas chromatography, chromatography with supercritical gas as mobile phase and electrophoretic separation processes.

10. Use of a kit according to any of claims 1 to 8 in nanoscale, microscale, analytical or preparative applications.

11. Use of a kit according to any of claims 1 to 8 in a continuously operated on-line separation technique and/or a separation process with an equilibrium mobile phase and/or a constant solvent separation process and/or an isothermal separation process.

12. Method for producing an optimized device for separating a substance mixture, characterized in that separating elements are coupled orConnected together, the separation element comprising a carrier element and the separation element comprising different stationary phases S_iWhere i.gtoreq.2, and each separating element T_iAll have a defined stationary phase S_iAnd of the same or different size, by the presence of, each stationary phase S_iWhere i.gtoreq.2, retention factor k determined by a prism model_iTo determine an optimal combination of selectivity and respective dimensions of the separation elements, and which are then joined or connected to form an optimal separation apparatus.

13. Method for producing an optimized device for separating substance mixtures, characterized by different stationary phases S_iWhere i.gtoreq.2, are linked together, linked together or coated on one another, by the presence of the stationary phases S_iThe stationary phase S is determined by the retention factor previously determined by the prism model_iAnd their size or volume, which are then linked together, optionally in a suitable carrier element, or coated with each other.