WO2004111634A1

WO2004111634A1 - System for thin-layer chromatography

Info

Publication number: WO2004111634A1
Application number: PCT/DE2003/000635
Authority: WO
Inventors: Thomas Raul Appel; Jevgenij Raskatov; Jan Rossa
Original assignee: Thomas Raul Appel; Jevgenij Raskatov; Jan Rossa
Priority date: 2002-02-26
Filing date: 2003-04-07
Publication date: 2004-12-23
Also published as: DE10209159A1; DE10209159B4

Abstract

The invention relates to a system for improving thin-layer chromatographic analysis, especially of chemical substances and molecular components of organisms. The system consists of the following array of devices: one or two thin-layer chromatography plates on which liquid or dissolved samples are applied manually with the aid of pipettes, syringes, cannulas or capillaries; vertical development chambers containing solvent or a solvent mixture; linings in the above-mentioned vertical chambers with an absorbent material for enriching the interior space of the vertical chambers with solvent vapors; retaining bows for fixing the absorbent material to the inner walls of the vertical chambers; development sections for mechanical and/or gravity-caused holding of the thin-layer chromatography plate(s) at a angle of less than 45 degrees relative to the perpendicular line, which is suitable for introducing the thin-layer chromatography plate(s) into the vertical chambers and for dipping in liquids; a heat stand for mechanical and/or gravity-caused mounting of one or two thin-layer chromatography plate(s) while minimizing heat-conductive bridges; a drying chamber or oven for regular heat treatment of the thin-layer chromatography plate(s) on the heat stand; application templates for exact and reproducible positioning of the pipettes, syringes, cannulas or capillaries during manual application of the samples.

Description

System to improve thin layer chromatographic analysis

description

The invention relates to a system for improving thin-layer chromatography analysis, in particular for the analysis of chemical substances and molecular components of organisms such as animals, plants, bacteria, fungi and viruses.

The thin polarity analysis uses the different polarity of substances / molecules.

A stationary separating agent fixed on the thin layer chromatography plate interacts with the dissolved substances to be separated depending on their properties. As a result of this and due to the different solubility of the substances in the eluent, the substances cover distances of different lengths on the separation layer of the thin layer chromatography plate. After coloring, substances can be determined qualitatively by assigning the running distance and also quantitatively by densitometry.

A single or a system of different solvents or solvent mixtures can be used to develop the thin-layer chromatography plates. Multiple development serves to focus the gangs and improve separation.

Separated substances (bands) can be made visible by spraying with or dipping in a colorant. In In some cases, subsequent even heating is required.

A qualitative evaluation is made by comparing the running distance and type of coloring with known substances. Reference values are needed for a quantitative determination. For this, the substance to be analyzed must be at the same concentration in several known concentrations

Thin layer chromatography plate can be applied as the sample. Densitometric evaluation, i.e. Comparison of the color intensities between the sample and controls enables a quantitative determination of the amount of substance contained in the sample. Since the intensity of the coloring can vary somewhat for each plate, these reference values must be reapplied to each thin-layer chromatography plate (H. Jork, W. Funk, W. Fischer, H. Wimmer: “Thin-layer chromatography, volume 1a, physical detection methods: basics , Reagenzien I .; VCH Verlagsges., Weilheim-Basel-Cambridge-New York, 1989).

It is known that vertical chambers with eluents or mixtures of different eluents and

Thin-layer chromatography plates, for example glass plates coated with the separating agent silica gel, can be used for material separation due to physical interactions (H. Jork, W. Funk, W. Fischer, H. Wimmer: "Thin-layer chromotography, Volume 1a, physical detection methods: basics, reagents I .; VCH Verlagsges., Weilheim-Basel-Cambridge-New York, 1989).

The high-resolution thin-layer chromatography on silica gel thin-layer chromatography plates enables, for example, the separation of all polar lipid classes (Touchstone JC 1995. Thin-layer chromatography procedures for lipid separation. J. Chromatogr. B 671: 169-95). In this context it is also known that a lining of the vertical chambers Filter paper can be used to saturate the chamber atmosphere with eluent.

For example, US Pat. No. 6,096,205 discloses a thin-layer chromatography system with analysis unit, which uses a multiplicity of supported thin-layer chromatography plates in a multiplicity of chambers and a CCD camera, so that a large number of samples can be examined simultaneously.

The application of a large number of samples to be analyzed on thin-layer chromatography plates by dabbing is known, for example, from US Pat. No. 4,272,381.

An expedient arrangement of the above devices is specified, which enables fast, accurate, robust, reproducible and inexpensive thin-layer chromatography analysis.

The essence of the invention is that the system for improving thin-layer chromatography analysis consists of an application template, a development frame, one or more holding sheets and a heating frame, which can be assigned to a thin-layer chromatography plate and one or more vertical chamber (s) with solvent / solvent mixture, in that the application template allows samples and controls to be applied at exact intervals on the thin-layer chromatography plate, the development frame enables safe and easy handling of the thin-layer chromatography plate during the

Chromatography and detection processes inside and outside the chamber make it possible for the holding sheets not to let the filter paper, which serves to saturate the atmosphere, slide off the walls of the chamber by holding it on the wall, and the heating frame for error-free heat treatment of the thin-layer chromatography plates outside the chamber without annoying thermal bridges.

The invention is explained in more detail below with reference to the figures and exemplary embodiments. 1 shows a schematic illustration of a system according to the invention,

2: an application template according to FIG. 1 in a sectional view,

Fig. 3: a development frame according to Fig. 1, Fig. 4: a tie according to Fig. 1 and

Fig. 5: a heating frame.

The system according to the invention for improving thin-layer chromatography analysis consists, as shown in FIG. 1, of one or more vertical chamber (s) 1, a thin-layer chromatography plate 2 mounted perpendicular to the bottom of the vertical chamber (s) 1, eluent or solvent mixture 3, which (s) the bottom of the vertical chamber (s)

1 and the bottom part of the thin-layer chromatography plate 2, a lining with absorbent material 4, which is mounted on the wall (s) of the vertical chamber (s) 1 and extends into the solvent or solvent mixture 3, an application template 5, a development frame 6 , one or more holding sheets 7 for fastening the absorbent material to the wall (s) of the vertical chamber (s) 1, and a heating frame 8, shown in more detail in FIG. 5, the application template 5, the development frame 6 and the heating frame 8 of the thin-layer chromatography plate 2 and the tie 7 of the vertical chamber 1 and the lining with absorbent material 4 can be assigned by the application template 5 an application of samples and controls at exact intervals on the thin layer chromatography plate

2 enables the development frame 6 to securely and easily hold the thin-layer chromatography plate 2 inside and outside the vertical chamber 1, the support bow 7 holds the lining with absorbent material 4 on the inner wall of the vertical chamber 1 in a non-slip manner and the heating frame 8 holds the thin-layer plate 2 outside the vertical chamber 1 for heat treatment without annoying thermal bridges.

The samples and controls are applied to the thin-layer chromatography plate 2 using a pipette, a syringe with a cannula (for example a Hamilton syringe) or a glass capillary, the application template 5 shown in FIG. 1 being designed with precisely defined recesses 51 which are designed as bores can be provided for the cannula or capillary receptacle, has an outer edge 52 as a spacer, so that when the application template 5 is placed on the thin-layer chromatography plate 2, there is a precisely fitting border and parallel spacing thereof, and thus manual application in precisely defined manner Distances is allowed.

This has the advantage that, in contrast to manual application without a template, an exact quantitative evaluation is possible by scanning individual webs with the same spacing.

The application template 5 consists of a transparent material, for example of plastic or glass, and is provided from above with recesses 51 / bores suitable for the Hamilton syringes or glass capillaries at regular intervals. The channel-like ones

Recesses 51 / bores are additionally tapered on the side facing away from the thin-layer chromatography plate 2 in order to make it easier to insert the syringes / capillaries. To identify the individual tracks, the recesses 51 / holes in the application template can be provided with numbers. 2 shows the shape of the recesses 51 / bores, which are conically recessed at the top of the application template 5 and pass through the application template 5 in the form of a channel downwards.

In addition, FIG. 2 shows the shape of the outer edge 52, which is the distance between the application template 5

Thin layer chromatography plate 2 adjusts and the thin layer chromatography plate 2 fits snugly on the side.

The vertical chamber (s) 1 is / are for better saturation of the atmosphere with the solvent / solvent mixture 3 with

Lining provided with absorbent material 4, whereby for

Fixation of the lining with absorbent material 4 in /

Fig. 4 illustrated (n) tie 7 find use. The purpose of the retaining bow 7 in the vertical chamber 1 is to fix the lining with absorbent material 4 in the vertical chamber 1 on the inner walls thereof.

3 shows the development frame 6, which serves to hold and hold the thin-layer chromatography plate 2 to facilitate handling inside and outside the vertical chamber (s) 1. The development frame 6 has two supports at the lower end for receiving thin-layer chromatography plates and has a bow-shaped shape at the upper end, so that the development frame 6 can be easily grasped by hand. In the upper third of the development frame 6 there is a holding tab, for example made of metal, which tilts those held by the development frame 6

Prevent thin layer chromatography plate (s) 2.

It is essential that the development frame 6 fits exactly into the interior of the vertical chamber 1 and holds the thin-layer chromatography plate 2 mechanically and / or by gravity at an angle of less than forty-five degrees to the vertical. This has the advantage that, in particular if development is to be carried out in succession with several eluents or if the thin-layer chromatography plate 2 is “washed” before development, the thin-layer chromatography plate 2 does not have to be touched with hands or objects when it is converted into different vertical chambers 1

During the final dive the

Thin-layer chromatography plate 2 in a colorant, which can also be located in a vertical chamber 1, for example, ensures that the development frame 6 securely and uniformly immerses the thin-layer chromatography plate 2 in the dye bath.

Both the development frame (s) 6 and the retaining arch (s) 7 are made of organic or inorganic materials (for example plastic, glass or metal), the connection of the individual elements from which the development framework 6 or retaining arch 7 are composed Spot welding or other connecting processes can be carried out. Machining of the development frame 6 or retaining bow 7 from solid material or shaping by casting or hardening from the liquid phase are also possible.

The heating frame 8 according to the invention, which is shown in one embodiment in FIG. 5, serves for easy handling of the thin-layer chromatography plate 2 for the visualization of latent colorations on the thin-layer chromatography plate 2 by uniform heating while avoiding undesired thermal bridges.

The embodiment of heating frame 8 consists of a metal frame, for example steel or aluminum, with six support fingers 81 pointing upwards. It offers a holder corresponding to the dimensions of the thin-layer chromatography plates used (for example 20 × 10 cm). The six support fingers 81 - four at the corners and two in the middle of the heating frame 8 - allow both 10 x 10 cm and 20 x 10 cm thin-layer chromatography plates to be used. The support fingers 81 limit the space available for the thin-layer chromatography plate 2 and thus prevent it from slipping. The thin-layer chromatography plate 2 itself lies only on small tips on the top of the support fingers 81, so that undesired thermal bridges are reduced to a minimum.

The embodiment of heating frame 8 is provided on the support fingers 81 with support points for the thin-layer chromatography plate 2, which can be formed, for example, by milled recesses. These recesses serve to fix the thin-layer chromatography plate 2 and prevent it from slipping when forces act on its side. The thin-layer chromatography plate 2 is therefore only in a punctiform and non-slip manner on the support fingers 81.

Other designs made of organic or inorganic materials, such as plastic or glass, are possible. The shape of the heating part 8 can deviate from the above embodiment, as long as the basic principle of mechanical and / or gravity-related storage of one or two thin-layer chromatography plate (s) 2 on their underside or their top side and / or their corners and / or their outside sides while minimizing heat-conducting bridges is satisfied. LIST OF REFERENCES:

1 vertical chamber

2 thin layer chromatography plate

3 solvent / solvent mixture

4 Lining with absorbent material

5 application template

51 - recesses

52 - outer edge

6 Development frame γ tie

8 heating rack

81 - Rest finger

Claims

claims

1. System for improving thin-layer chromatographic analysis according to Figure 1 consisting of one or

5 several vertical development chamber (s) (1); one or two thin-layer chromatography plate (s) (2) stored at an angle of less than forty-five degrees to the vertical within the above vertical chamber (s) (1) (2); Solvent (s) or solvent mixture (s) (3), which (s) the bottom of the o vertical chamber (s) (1) and the lowest part of the

Cover thin layer chromatography plate (s) (2); Lining (s) inner walls of the vertical chamber (s) (1) with absorbent material (4), which is immersed in eluent or solvent mixture (s) (3), to enrich the 5 interior of the vertical chamber (s) (1) with eluent vapor ; one or more application template (s) (5) according to Figures 1 and 2 for the exact and reproducible positioning of pipettes, syringes, cannulas or capillaries for the purpose of manual application of liquid or dissolved o samples onto the thin layer chromatography plate (s) (2); one or more development frame (s) (6) according to Figure 3 for mechanical and / or gravity-related mounting of the thin-layer chromatography plate (s) (2) at an angle of less than forty-five degrees to the vertical, 5 suitable for the introduction of the

Thin-layer chromatography plate (s) (2) in the vertical chamber (s) (1) and for immersing the thin-layer chromatography plate (s) (2) in liquids; one or more holding bends (7) according to Figure 4 for fastening the lining (s) with absorbent material (4) to the inner walls of the vertical chamber (s) (1); a heating frame (8) according to Figure 5 for mechanical and / or gravity-related storage of one or two thin-layer chromatography plate (s) (2) on its underside 5 or its top and / or its corners and / or its

Outside while minimizing heat-conducting bridges. Thin-layer chromatography plate (s) (2), application template (s) (5), development frame (s) (6), retaining sheet (7) and heating frame (8) are the vertical chamber (s) (1) and the lining (s) assignable with absorbent material (4) by

Application template (s) (5) for the exact and reproducible positioning of pipettes, syringes, cannulas, capillaries or pipettes for the purpose of manual application of liquid or dissolved samples to the thin-layer chromatography plate (s) (2);

Development rack (s) (6) die

Hold thin-layer chromatography plate (s) (2) inside and outside of the vertical chamber (s) (1) in order to transfer the thin-layer chromatography plate (s) (2) from one easily and safely

Ensure vertical chamber (1) in another vertical chamber (1) and immersion of the thin-layer chromatography plate (s) (2) in liquids without bringing hands, tweezers or other aids into contact with the liquids; the retaining arch (7) holds the lining (s) with absorbent material (4) on the inner walls of the vertical chamber (s) (1) so that they do not slip; and holding the heating frame (8) outside the vertical chamber (s) (1) thin-layer chromatography plate (s) (2) for heat treatment without annoying thermal bridges.

2. Application templates for thin layer chromatography plates made of transparent material (for example glass or plastic) according to Figures 1 and 2, adapted to the

Format of the thin layer chromatography plates and provided with recesses at defined intervals. The recesses are used for the manual application of samples and controls by means of pipettes, syringes, cannulas or capillaries in defined, easy to maintain

Intervals and thus facilitate the exact quantitative evaluation. Application templates (5) are characterized in that the application template (5) on its side facing away from the thin-layer chromatography plate (2) has a conically countersunk, later vertically channel-like extending recesses (51) and on their side facing the thin layer chromatography plate (2) is provided with a raised outer edge (52), the recesses (51) having precisely defined diameters for the pipette, syringe, cannula or capillary holder and the like outer edge (52) a precisely fitting border and parallel spacing of the application template (5)

Thin layer chromatography plate (2) generated.

3. Development racks made of inorganic or organic material (for example metal, glass or plastic) according to Figure 3, for mechanical and / or gravity-related mounting of one or two thin-layer chromatography plates (2) at an angle of less than forty degrees to the vertical. Development racks (6) are characterized in that they are suitable for the intended introduction of one or two thin-layer chromatography plates (2) into vertical chambers (1) and for immersion of one or two

Thin-layer chromatography plates (2) in liquids without touching the liquids with hands, tweezers or other aids.

4. Retaining arches (7) made of inorganic or organic material (for example metal, glass or plastic) according to Figure 4, for fastening linings made of absorbent material (for example filter paper) to the inner walls of vertical chambers (1). Ties (7) are characterized in that they ensure the non-slip holding of the absorbent material on the inner walls of vertical chambers (1) by spring force or other pressing forces and at the same time do not hinder the introduction of development frames (6) into the vertical chambers (1).

5. heating frame (8) made of inorganic or organic

Material (e.g. metal, glass or plastic) is used to uniformly heat thin-layer chromatography plate (s) (2) in one Drying cabinet or oven for visualization of latent stains on thin layer chromatography plate (s) (2) while minimizing undesired thermal bridges. The heating frame (8) is characterized in that one or two thin-layer chromatography plates (2) are placed thereon while minimizing heat-conducting bridges and are stored mechanically and / or due to gravity on their underside or top side and / or their corners and / or their outside sides.

6. An embodiment of the heating frame (8) according to Figure 5 consists of a rectangular frame, which is provided with six support fingers (81) directed perpendicular to the frame. The described embodiment of the heating frame (8) is characterized in that four of the

Support fingers (81) at the corners of the frame and one support finger (81) opposite each other, positioned centrally between two support fingers (81) of the corners. The described support fingers offer one or two thin layer chromatography plate (s) (2) a suitable one

Bracket. A full-size thin-layer chromatography plate can be used with six support fingers

(e.g. 20 x 10 cm) as well as one or two thin-layer chromatography plate (s) half size (e.g. 10 x 10 cm). The support fingers (81) limit the support surface available for the thin-layer chromatography plates (2), for example by means of cut-out recesses, and thus prevent them from slipping sideways. The thin-layer chromatography plates (2) rest only in a punctiform manner on small tips at the corner (s) of the support fingers (81) positioned in each case towards the interior of the frame, so that undesired thermal bridges are reduced to a minimum.