FR2649796A1 - Improvement made to the developing step of thin-layer chromatography, developing device, and installation comprising the said device - Google Patents

Abstract

The method of developing in thin-layer chromatography comprises placing the chromatography plate to be developed in contact, under substantially uniform pressure, with a suitable material impregnated with a suitable developing substance or reactant. The developing device for thin-layer chromatography comprises: - a porous material 20, 20', which is resistant to normal chromatography developing reactants and has a sufficient absorbent power to completely impregnate a chromatography plate without immersing it at the moment of placing in contact under pressure and to reabsorb the excess reactant during withdrawal of the said porous material from the chromatography plate and - a handling support 10, 11; 10', 11' which is inert with respect to the developing reactants, of sufficient rigidity to allow the application of a suitable pressure onto a thin-layer chromatography plate and on which the said porous material is mounted or fixed.

Description

 The present invention relates to an improvement made in the step of revealing the thin layer chromatography, a revelation device for implementing said improvement as well as a.

installation comprising said device.

 Thin layer chromatography (TLC) is a method of differential migration separation between a stationary phase and a mobile phase. The thin layer has a number of advantages (including rapid migration, use of any type of revelation reagent).

 It is a method of identification which makes it possible in particular to determine the purity of a product, the decomposition products giving additional tasks.

 CCM has many qualitative applications in both the pharmaceutical and agri-food sectors. However, the CCM lends itself to quantitative interpretation with errors of 5 to more than 10% in some cases.

The CCM has five steps
the deposit of the substances to be identified and / or to be dosed on an appropriate support
. the migration
. drying
. the revelation
reading.

 A certain number of these operations have been optimized, in order to reduce the risks of errors inherent in the various manipulations; in particular, the operations of filing, migration, drying and reading have been automated in particular and have significantly reduced the risk of error; on the other hand, revelation; at the present time, when necessary, is carried out either by spraying specific reagents which make it possible to obtain colored or fluorescent stains or by total immersion of the chromatography plate.

These techniques of development by spraying or immersion have a number of disadvantages
In particular, the spraying
- gives different results depending on the manipulator
- gives a non-homogeneous background
- may require the use of compressed air, and therefore of a particular installation
- may cause streaks or flows that make reading difficult to interpret.

The immersion revelation for its part
- may cause diffusion and dilution of stains
- gives a background too colorful
- claims a significant amount of developer.

 It has therefore been the object of the present invention to provide an improvement of the thin layer chromatography revealing step, which is more relevant to the requirements of the practice than the disclosure methods heretofore employed, in particular in that it has the advantage of improving, unexpectedly, the reproducibility and repeatability of thin layer chromatography techniques, more particularly in their quantitative applications.

 It is also an object of the invention to provide a revelation device for implementing said improvement, as well as an installation comprising said device.

 The subject of the present invention is a process for developing thin-layer chromatography, characterized in that it comprises bringing the chromatography plate to come into contact with a suitable material impregnated with a substance or reagent of the same at a substantially uniform pressure. appropriate disclosure.

 For the purposes of the present invention, the term "pressure" means the action of pressing or pushing with force, said pressure being able to be exerted manually or mechanically.

 According to an advantageous embodiment of the invention, said suitable material is made of a porous material, resistant to the revelation reagents customary for the chromatography and having an absorbency sufficient to completely impregnate said chromatography plate, without the immerse during the contacting under pressure and to reabsorb the excess reagent, at the time of removal of said material from the chromatography plate.

 According to an advantageous arrangement of this embodiment, said porous material is a suitable polymer.

 Such a revelation process has the advantage of giving a uniform background and unexpectedly improving the reproducibility and repeatability of thin layer chromatography techniques, in particular quantitative techniques.

 Such a method also has the advantage of being easily automatable.

The present invention also relates to a revelation device for thin layer chromatography, characterized in that it comprises
a porous material which is resistant to the usual chromatographic reagents and which has sufficient absorbency to completely impregnate a chromatographic plate without immersing it at the time of contacting under pressure and to reabsorb the excess reagent during the removing said porous material from the chromatography plate, and
an inert handling support with respect to the revealing reagents, of sufficient rigidity to allow the application of an appropriate pressure on a thin-layer chromatographic plate and on which said porous material is mounted or fixed.

 According to an advantageous embodiment of said device, it comprises as porous material, a suitable polymer.

 Said polymer advantageously has a foam or sponge structure.

 According to an advantageous arrangement of this embodiment, said polymer is an open-cell polyurethane foam.

 According to another advantageous embodiment of said device, it is of cylindrical shape, for example a roller or has a convex semi-cylindrical surface.

 According to yet another advantageous embodiment of said device, it is of rectangular shape.

 Said device necessarily has a planar contact zone with the chromatography plate, to apply a substantially constant and uniform pressure on the chromatography plate, whatever the contact zone.

The present invention further relates to a suitable installation in the step of developing the thin layer chromatography, characterized in that it comprises
an impregnation zone of the revelation device according to the invention
a revealing zone where the thin layer chromatography plate is to be revealed
means for guiding and moving the device between a position where it is in the impregnation zone and a rest position, the displacement between these two positions causing the device according to the invention to be pressurized with the thin-layer chromatography plate and impregnation of said plate with the revealing reagent.

 According to an advantageous embodiment of the installation, it further comprises a dewatering zone of the disposotif, between the impregnation zone and the revelation zone.

 According to the invention, said device is advantageously a roll of cylindrical shape, comprising an axial shaft whose ends are formed in guide rails extending laterally on either side of the aforementioned areas of impregnation, revelation and rest.

 According to another advantageous embodiment of the installation according to the invention, it is in the form of a box with pivoting lid, allowing access to the aforementioned areas of impregnation, revelation and rest and delimiting the aforementioned slides with the fixed side walls of said box.

 In addition to the foregoing, the invention also includes other provisions, which will emerge from the description which follows.

The invention will be better understood with the aid of the additional description which follows, which refers to examples of implementation of the method according to the invention and to a detailed description of the revelation device and the installation according to the invention. the invention with reference to the accompanying drawings in which
FIG. 1 is a perspective representation of a rectangular embodiment of the device according to the present invention;
FIG. 2 is a representation of another rectangular embodiment of the device according to the invention;
FIG. 3 is a representation of a cylindrical embodiment of the device according to the invention;
- Figures 4, 5 and 6 are a representation of an embodiment of the installation according to the invention: Figures 4 and 5 are sectional views and Figure 6 is a perspective view.

 It should be understood, however, that these drawings and the corresponding descriptive parts and examples are given solely by way of illustration of the subject of the invention, and in no way constitute a limitation thereof.

xemp1e. i
Thin layer chromatography for the tryptophan assay is carried out as follows - plates: non-fluorescent cellulose 20 x 20 cm (MERCK) - mobile phase: Butanol 1: 80 volumes
Acetic acid: 20 volumes
Water: 100 volumes - samples: tryptophan solution at 2 mg / ml - deposits: 10 deposits of 2 μl, made at
using LINOMAT IV (CAMAG) - revealing reagent: ninhydrin / copper
solution A: ninhydrin: 1.6 g
ethanol: 800 ml
acetic acid: 160 ml
2-4-6 collidine: 32 ml
solution B: cupric nitrate at 2 ° in ethanol
100 ml of solution A and 6 ml of solution B are mixed to obtain the revealing reagent.

A revelation device according to the invention is impregnated with the revelation reagent above
said device imbibed under pressure is applied to the chromatography plate at the time of the revelation;
the photodensitometric reading is performed using a CD60 device (DESAGA). The plates are read before and after revelation using the device according to the invention.

1. Chromatogramam not revealed - reading at 254 nm (direct UV):

<tb> DEPOT <SEP> AREA <SEP> HEIGHT
<tb> 1 <SEP> 789.1 <SEP> 173.48
<tb> 2 <SEP> 637.8 <SEP> 143.91
<tb><SEP> 3 <SEP> 715.5 <SEP> 117.09
<tb><SEP> 4 <SEP> 827.0 <SEP> 193.25
<tb><SEP> 5 <SEP> 888.4 <SEP> 205.85
<tb><SEP> 6 <SEP> 859.4 <SEP> 205.44
<tb><SEP> 7 <SEP> 879 ,! <SEP> 209.75
<tb> 8 <SEP> 873.1 <SEP> 203.51
<tb><SEP> 9 <SEP> 897.7 <SEP> 202.95
<tb> 10 <SEP> 874.5 <SEP> 182.27
<tb><SEP> m <SEP> = <SEP> 824.16 <SEP> m <SEP> = <SEP> 188.75
<tb><SEP>#<SEP><SEP> = <SEP> 85.958 <SEP>#<SEP> = <SEP> 21.619
<tb><SEP> CV <SEP> = <SEP> 10.43 <SEP>% <SEP> CV <SEP> = <SEP> 11.45 <SEP>%
<Tb>
Repeatability is measured by the coefficient of variation (CV); it is even better than the coefficient of variation is weak.

 2. Chromatogram after ninhydrin revelation by the device. revelation under pressure -.

reading at 510 nm:

<tb> DEPOT <SEP> AREA <SEP> HEIGHT
<tb><SEP> 1 <SEP> 2312.9 <SEP> 460.27
<tb><SEP> 2 <SEP> 2136.7 <SEQ> 428.67
<tb><SEP> 3 <SEP> 2225.0 <SEQ> 467.70
<tb><SEP> 4 <SEP> 2268.3 <SEQ> 482.02
<tb><SEP> 5 <SEP> 2209.7 <SEP> 471.81
<tb><SEP> 6 <SEP> 2122.8 <SEQ> 460.02
<tb><SEP> 7 <SEP> 2142.7 <SEP> 465.76
<tb><SEP> 8 <SEP> 2174.3 <SEP> 452.84
<tb><SEP> 9 <SEP> 2188.1 <SEP> 455.28
<tb> 10 <SEP> 2207.4 <SEP> 422.76
<tb><SEP> m <SEP> = <SEP> 2198.8 <SEP> m <SEP> = <SEP> 456.71
<tb><SEP> o <SEP> = <SEP> 59,835 <SEP> a <SEP> = <SEP> 18,411
<tb><SEP> CV <SEP> = <SEP> 2.72 <SEP>% <SEP> CV <SEP> = <SEP> 4.03 <SEP>%
<Tb>
The disclosure device according to the invention makes it possible, unexpectedly, to obtain significantly improved results and accuracy compared with the reference technique (UV).

Example 2
The results obtained in Example 1 are compared with those obtained when, under the same conditions, the revelation is carried out by spraying.

1. Chromatogram not revealed - reading at 254 nm (direct UV):

<tb> DEPOT <SEP> AREA <SEP> HEIGHT
<tb> 1 <SEP> 922.1 <SEP> 181.59
<tb> 2 <SEP> 918.1 <SEP> 179.51
<tb><SEP> 3 <SEP> 869.5 <SEP> 175.36
<tb><SEP> 4 <SEP> 871.4 <SEP> 170.30
<tb><SEP> 5 <SEP> 842.5 <SEP> 167.04
<tb><SEP> 6 <SEP> 833.9 <SEP> 164.93
<tb><SEP> 7 <SEP> 835.3 <SEP> 166.51
<tb><SEP> 8 <SEP> 864.8 <SEP> 171.63
<tb><SEP> 9 <SEP> 829.6 <SEP> 170.97
<tb> 10 <SEP> 870.8 <SEP> 174.08
<tb><SEP> m <SEP> = <SEP> 865.8 <SEP> m <SEP> = <SEP> 172.19
<tb><SEP> a <SEP> = <SEP> 32.96 <SEP> a <SEP> = <SEP> 5,502
<tb><SEP> CV <SEP> = <SEP> 3,81 <SEP>% <SEP> CV <SEP> = <SEP> 3,19 <SEP>%
2. Chromatogram after ninhydrin revelation by conventional spraying - reading at 510 nm.

<Tb>

DEPOT <SEP> AREA <SEP> HEIGHT
<tb> 1 <SEP> 1686.9 <SEP> 321.07
<tb> 2 <SEP> 1639.0 <SEP> 312.03
<tb><SEP> 3 <SEP> 1729.3 <SEP> 331.35
<tb> 4 <SEP> 1743.1 <SEP> 332.25
<tb><SEP> 5 <SEP> 1676.3 <SEP> 327.02
<tb><SEP> 6 <SEP> 1614.7 <SEP> 313.29
<tb><SEP> 7 <SEP> 1508.7 <SEP> 290.53
<tb> 8 <SEP> 1455.4 <SEP> 283.81
<tb> 9 <SEP> 1493.4 <SEP> 296.73
<tb> 10 <SEP> 1482.8 <SEP> 290.04
<tb><SEP> m <SEP> = <SEP> 1602.96 <SEP> m <SEP> = <SEP> 309.812
<tb><SEP> a <SEP> = <SEP> 108.81 <SEP> a <SEP> = <SEP> 18.309
<tb><SEP> CV <SEP> = <SEP> 6.79 <SEP>% <SEP> CV <SEP> = <SEP> 5.91 <SEP>%
<Tb>
When using the conventional revelation technique, by spraying, the results are worse than in direct UV.

 The process according to the invention may in particular be carried out using one of the devices described below.

 The device according to the present invention, shown in Figure 1 comprises a rigid support 10 provided with a handling member (gripping) 11, on which is mounted a polymer material 20, soaked with revealing reagent. In this embodiment, and in a nonlimiting manner, said device is square in shape, with a side of 200 mm, that is to say of the same size as the chromatography plates; the polymeric material has a thickness of 4.5 mm, while the rigid support has a thickness of 20 mm.

Said device has a sectional shape
T inverted and is advantageously used to apply the revealing reagent to a chromatography plate, as described in Example 1 above.

 After use, said device is stored in suitable hermetic tanks.

 FIG. 2 represents another embodiment of the device according to the invention in which the portion of the rigid support 10 in contact with the polymeric support 20 soaked in reagent is slightly curved.

 The device according to the present invention, shown in Figure 3. comprises a rigid support 10 'in the form of a roller, provided with a handling axis 11' around which is applied the polymeric porous material 20 ', soaked with revelation reagent as described in Example 1 above.

 Said device is used in particular in an installation according to the invention, as can be seen in FIGS. 4, 5 and 6, for applying the developing reagent by rolling on a chromatography plate 30.

FIG. 4 represents an embodiment of an installation according to the invention in the form of a box 2, which comprises
an impregnation zone A of the device 1, represented in this installation by a tank in which there is a developing reagent, as described in Example 1
a revealing zone C, where the chromatography plate 30 to be revealed is located, and which also comprises the plate supports 311 and 312 as well as the stops 32 and 322, which block said plate
means for guiding and displacing the device 1 between a position where it is in the impregnation zone and a so-called rest position (zone
D); the ends of the axis 11 'of said device are thus received in the slides 41 of the means 40 extending over the chromatography plate 30
- A dewatering zone B of the device 1 (position lb of said device), located between the impregnation zone A and the revealing zone C.

 Such an installation allows the application of a constant and uniform pressure on said plate 30 and thus makes it possible to obtain a clearly improved revelation, as shown in Examples 1 and 2 above.

 A device according to the invention 1 is first placed in the impregnation zone A, in position la and is thus impregnated with revealing reagent; then said device is transferred in position 1b, in the dewatering zone B, in which any excess reagent is drained, before the device 1 passes into the revealing zone C, in which said reagent is released from the device and evenly impregnates the plate to reveal.

 FIG. 5 represents said installation, with the removable part 50a of the pivoting cover 50, which allows access to the open impregnation and revealing zones. Said cover has two side edges d cut in steps, said flanges of the cover defining the slideway 41 of the means 40 with the walls. 60 side of the box 2, cut to correspond to said flanges.

 FIG. 6 represents a perspective view of the installation as described in FIGS. 4 and 5.

 As is apparent from the above, the invention is not limited to those of its modes of implementation, implementation and application which have just been described more explicitly; it encompasses all the variants that may come to the mind of the technician in the field, without departing from the scope or scope of the present invention.

Claims (7)

 1 ') revealing process in thin layer chromatography, characterized in that it comprises a contacting under a substantially uniform pressure of the chromatography plate to be revealed with a suitable material, impregnated with a substance or appropriate revelation reagent .  2-) Process according to claim 1, characterized in that said suitable material is made of a porous material, resistant to the revelation reagents customary chromatography and having sufficient absorbency to completely impregnate said chromatography plate, without the immerse during the contacting under pressure and to reabsorb the excess reagent, at the time of removal of said material from the chromatography plate.  3) Process according to claim 1 or claim 2, characterized in that said porous material is a suitable polymer.  a handling support (10, 11; 10 ', 11') which is inert with respect to the revealing reagents, of sufficient rigidity to allow the application of an appropriate pressure on a thin-layer chromatographic plate and on which is mounted or fixed said porous material.  a porous material (20, 20 ') which is resistant to the usual chromatographic reagents and has sufficient absorbency to completely impregnate a chromatographic plate without immersing it at the time of pressurized contact and to reabsorb the excess reagent upon removal of said porous material from the chromatography plate, and  4) Disclosure device for thin layer chromatography, characterized in that it comprises  Device according to Claim 4, characterized in that it comprises, as porous material, a suitable polyamine.  6 ') Device according to claim 5, characterized in that said polymer is an open-cell polyurethane foam.  8 ') Device according to any one of claims 4 to 6, characterized in that it is rectangular in shape.  7 ') Device according to any one of claims 4 to 6, characterized in that it is cylindrical in shape, such as a roller or has a convex semi-cylindrical surface.  means for guiding and displacing the device between a position where it is in the impregnation zone and a rest position, the displacement between these two positions causing the device to be pressurized according to FIG. any one of claims 4 to 8 with the thin layer chromatographic plate and impregnation of said plate with the developer reagent.  a revealing zone (B) where the thin-layer chromatography plate (30) is to be revealed  an impregnation zone (A) of the developing device (1) according to any one of claims 4 to 8  9 ') Suitable installation in the step of revealing the thin layer chromatography, characterized in that it comprises:  10 ') Installation according to claim 9, characterized in that it further comprises a dewatering zone (B) of the device, between the impregnation zone and the revelation zone.  -11 ') Installation according to claim 9 or claim 10, characterized in that said device 1 is preferably a cylindrical roller, comprising an axial shaft whose ends are received in guide rails (41) extending laterally on both sides of the aforementioned zones of impregnation, revelation and rest.  12 ') Installation according to any one of claims 9 to 11, characterized in that it is in the form of a box (2) with cover (50) whose pivoting portion (50a) allows access to aforementioned areas of impregnation, revelation and rest and delimiting the aforementioned slides with the fixed side walls (60) of said box 2.