Patent Application
20120164744
The present invention relates to a method of carrying out a three-dimensional chromatography according to the preamble of claim 1.
The principle of chromatography as such has been known from the prior art for a long time. Here, a mobile phase containing (specific) components to be separated passes through columns, for example. Specific interactions of the analytes with the stationary phase lead to a separation of the individual components emerging from the column with different time lags. For a separation of highly complex samples/mixtures, two-dimension chromatography outperforms one-dimensional chromatography, and three-dimensional chromatography in turn outperforms two-dimensional chromatography.
The principle of three-dimensional chromatography is known from document U.S. Pat. No. 4,469,601, too. There, a two-dimensional chromatography is performed along a plate. The plate is permeable and, when dried, is placed against a cube of material. Then, a solvent is forced through the plate and through the cube in order to use the retention mechanism that is provided in that third dimension. Although this way of proceeding of the three-dimensional chromatography is troublesome and the structure of the separation body is complex, the method as such nevertheless represents the advantages of spatial (3D) chromatography.
Usually, the mobile phase migrates through the separation body as a result of a pressure difference applied. Alternatively, under suitable conditions an (electro-osmotic) flow may also be realised by applying an electric voltage.
In the light of this known prior art, the object of the present invention is to improve the results of three-dimensional chromatography with suitable means leading to better and more reliable (precise, accurate) results in a shorter time.
The object is achieved with a method according to claim 1.
The invention is based on the idea that the course of the three-dimensional chromatography is essentially influenced by the way the mobile phase moves through the separation body and that it is possible to use suitable means to influence the flow behaviour of the mobile phase inside the three-dimensional separation body.
The method according to the invention starts out from a three-dimensional separation body which extends in space in three directions X, Y and Z preferably orthogonal to one another. The three-dimensional chromatography is preferably carried out in three successive steps; in each step, a mobile phase is forced through the separation body in one of the directions X, Y or Z. The retention capacity of the separation body for sample components transported with the mobile phase causes their separation along this flow direction X, Y or Z. Every subsequent step of chromatography leads to a further separation of those components which have already been separated in a direction perpendicular thereto in the preceding step. The third and last step leads to a distribution or separation of the components in the Z-direction (separation in space) or to a temporal separation of the components, with the result that they emerge from the separation body at a boundary surface in the Z-direction at different points of time (separation in time).
As already mentioned, the mobile phase is typically caused to pass through the separation body by applying a pressure difference or an electric voltage.
According to the invention, this method, which is known as such, is developed further in that the flow of the at least one mobile phase inside the separation body is deliberately controlled as to space and/or time by locally influencing the chemical and/or physical properties of the separation body. Through this, the flow of the mobile phase inside the separation body is controlled or restricted deliberately and in a locally dependent manner, so a more precise result of chromatography can be obtained by means of more exactly predetermined flows. In particular, with the aid of this influence, the flow can be optimally restricted to precisely one of the directions X, Y or Z, so spreading of the mobile phase transversely to this deliberately caused direction of flow is prevented in the best way possible. Thereby, uncertainties in the developments achieved can be reduced or avoided completely.
Moreover, locally influencing the physical or chemical properties of the separation body also makes it possible to control the flow along the entire direction X, Y or Z. Thus, it would also be possible to cause different flow rates within one step of chromatography, i.e. along one of the directions X, Y or Z, so the mobile phase flows partially faster, partially more slowly, in that particular direction.
The flow control according to the invention can be promoted by providing zones of different chemical or physical properties inside the separation body from the start, so the mobile phase is automatically subjected to the local flow conditions and is guided accordingly along the flow path it takes. Another aspect of the invention consists in deliberately influencing the physical or chemical properties inside the separation body in terms of space or time by means of external action so as to be able to design the flow conditions inside the separation body.
An embodiment of the method according to the invention provides that the direction of flow and/or amount of flow inside the separation body is controlled by local electromigration by controlling the surface charge.
Another aspect of the invention is directed to eluent demixing and dewetting phenomena. Here, flow control is achieved by restraining the mobile phase to specific areas of the separation body.
Another aspect of the invention relates to chemical reagents, which may only be made available in certain regions of the separation body, for example by allowing the reagent to diffuse into the body by capillary action initiated from outside the body during a limited period of time. Alternatively, any of the flow control options, as described in this patent, can be used to deliver reagents to specific parts of the separation body.
Another way to control the flow inside the separation body is to activate micro- or nanoswitches in order to open or close pores or channels inside the body to allow or obstruct flow of the eluent. Those switches may be activated by applying a voltage or chemical reaction with reagents introduced into the mobile phase. Alternatively, such switches may be activated by radiation focused on certain areas of the body.
In general, flow control according to the invention may advantageously be performed over a limited period of time or within a limited area of the separation body. Thus, temporary flow control may serve to e.g. permit the flow of the mobile phase only in the X-direction for the moment. Then, the influencing factors determining the direction of flow may be cancelled or changed in such a way that the mobile phase now basically flows in the Y-direction or in the Z-direction. Independently of or in addition to the outer conditions (pressure difference or electric voltage applied), the flow of the mobile phase inside the block may thus be directed to a specific direction X, Y or Z by means of well-aimed influence from outside. In particular, depending on the type of influence from outside, the flow of the mobile phase may be directed into different directions X, Y or Z of space at a specific position inside the separation body in order to obtain a well-aimed flow and above all in order to avoid a flow of the mobile phase into a different direction that is not desired at this point of time. On the one hand, this makes it possible to accelerate the three-dimensional chromatography to a considerable extent and on the other hand, the individual target components can be separated from one another more clearly since the flow of the mobile phase can be restricted to only one direction of space for each step of chromatography in the best way possible.
Other advantageous embodiments are defined in the subclaims.
In the following, the basic principle of the method according to the invention is to be explained in greater detail with the aid of an example shown in the FIGURE.
As shown in
To perform three-dimensional chromatography, the mobile phase is first introduced to the body through a limited upper edge area 2, which is illustrated in
During this first step, a separation of components along the X-axis of the block will occur according to the retention mechanism provided in that direction. Preferably, the components will distribute individually along that first axis (“separation in space”).
After this first step, a mobile phase is introduced to penetrate the upper edge of the body 2 in the Y-direction, perpendicular to the distribution of the first step and along the entire length X through a narrow strip 3. The phase will flow in the Y-direction, preferably without any variations into the other directions X or Z, and effect an additional separation in this second dimension of those components, which were located at a specific X-position after the first step. As a result, the component will further be separated in another “separation in space” across an X-Y-area, which could be the upper surface of the body 1 of
The third separation step includes penetration of the body perpendicular to the X-Y-surface that has undergone the previous step. A mobile phase is forced through the body in the Z-direction, causing another separation of the components which were located at specific X-Y-positions after the second step of the three-dimensional chromatography.
This separation may again occur “in space”, ending up with a distinct distribution of components along all three directions X, Y and Z of the body. Another type of separation (“in time”) occurs when the components are driven through the body entirely for this last step, but emerge from it at different points of time due to the retention mechanism chosen in that Z-direction.
According to the invention, the flow of the mobile phase inside the separation body is deliberately controlled in terms of space and/or time by locally influencing the chemical and/or physical properties of the separation body.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2009 040 180.6 | Sep 2009 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP10/05468 | 9/7/2010 | WO | 00 | 3/6/2012 |
