CA2035025C - Device and installation of thin film chromatography, with improved development - Google Patents
Device and installation of thin film chromatography, with improved development Download PDFInfo
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- CA2035025C CA2035025C CA002035025A CA2035025A CA2035025C CA 2035025 C CA2035025 C CA 2035025C CA 002035025 A CA002035025 A CA 002035025A CA 2035025 A CA2035025 A CA 2035025A CA 2035025 C CA2035025 C CA 2035025C
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
Improvements have been made to the developing step in thin layer chromatography, both on a developing device for carrying out the improvement, and in apparatus containing the device.
The improvements are on a developing method in a thin-layer chromatography developing procedure in which a chromatographic plate to be developed is brought into contact with a material which is impregnated with a developing reagent. The improvement includes pressing, in a substantially-uniform manner, a porous polymer containing the developing reagent, against the thin-layer chromatography plate to be developed.
The porous polymer possesses sufficient absorbing power completely to impregnate the chromatographic plate without immersing it during the contact under pressure, and a capacity for reabsorbing excess reagent when the porous polymer is withdrawn from the plate. The developing device for the thin layer chromatography comprises a porous polymer which is resistant to conventional chromatography developing reagents and which has sufficient absorbing power completely to impregnate a chromatographic plate without immersing it at the time of contact under pressure and to reabsorb excess reagent when the porous material is withdrawn from the chromatographic plate. The device includes a manipulation support which is inert with respect to the developing reagents, and which has sufficient rigidity to allow an appropriate pressure to be applied to the thin-layer chromatographic plate and on which the porous material is mounted or fixed.
The improvements are on a developing method in a thin-layer chromatography developing procedure in which a chromatographic plate to be developed is brought into contact with a material which is impregnated with a developing reagent. The improvement includes pressing, in a substantially-uniform manner, a porous polymer containing the developing reagent, against the thin-layer chromatography plate to be developed.
The porous polymer possesses sufficient absorbing power completely to impregnate the chromatographic plate without immersing it during the contact under pressure, and a capacity for reabsorbing excess reagent when the porous polymer is withdrawn from the plate. The developing device for the thin layer chromatography comprises a porous polymer which is resistant to conventional chromatography developing reagents and which has sufficient absorbing power completely to impregnate a chromatographic plate without immersing it at the time of contact under pressure and to reabsorb excess reagent when the porous material is withdrawn from the chromatographic plate. The device includes a manipulation support which is inert with respect to the developing reagents, and which has sufficient rigidity to allow an appropriate pressure to be applied to the thin-layer chromatographic plate and on which the porous material is mounted or fixed.
Description
(a) TITLE OF THE INVENTION
DEVICE AND INSTALLATION OF THIN FILM CHROMATOGRAPHY, WITH
IMPROVED DEVELOPMENT.
(b) TECHNICAL FIELD TO WHICH THE INVENTION RELATES
The present invention relates to an improvement made to the developing step in than Layer chromatography, to a developing device for carrying out the improvement, as well as to an apparatus containing that device.
(c) BACKGROUND ART
Thin layer chromatography (TLC) is a method of separation by differential migration be-~ween a stationary phase and a mobile phase. The thin layer offers a certain number of advantages (especially speed of migration, and the use of any type of developing agent).
It is an identification rnethod which makes it possible, in particular, for the purity o:E a product to be determined, the decomposition products giving rise to additional spots.
TLC offers numerous qualitative applications in the pharmaceutical field as well as in the food technology field.
Nevertheless, TLC lends itse_Lf to quantitative interpretation with errors of 5 to more than loo in some cases.
TLC generally comprises five steps namely, depositing of the substances to be identified and/or determined on a suitable support; migration; drying; developing; and reading.
A :number of these operat__ons have been optimised, in order to reduce the risk of errors inherent in the different manipulations. In particular,. the depositing, migration, drying and reading operations have especially been automated and have markedly reduced the risk of errors. On the other hand, developing, at the present time, when it is necessary, is carried out either by spraying of specific reagents which allow coloured or fluorescent. spots to :be obtained, or by total immersion of the chromatographic :plate.
DEVICE AND INSTALLATION OF THIN FILM CHROMATOGRAPHY, WITH
IMPROVED DEVELOPMENT.
(b) TECHNICAL FIELD TO WHICH THE INVENTION RELATES
The present invention relates to an improvement made to the developing step in than Layer chromatography, to a developing device for carrying out the improvement, as well as to an apparatus containing that device.
(c) BACKGROUND ART
Thin layer chromatography (TLC) is a method of separation by differential migration be-~ween a stationary phase and a mobile phase. The thin layer offers a certain number of advantages (especially speed of migration, and the use of any type of developing agent).
It is an identification rnethod which makes it possible, in particular, for the purity o:E a product to be determined, the decomposition products giving rise to additional spots.
TLC offers numerous qualitative applications in the pharmaceutical field as well as in the food technology field.
Nevertheless, TLC lends itse_Lf to quantitative interpretation with errors of 5 to more than loo in some cases.
TLC generally comprises five steps namely, depositing of the substances to be identified and/or determined on a suitable support; migration; drying; developing; and reading.
A :number of these operat__ons have been optimised, in order to reduce the risk of errors inherent in the different manipulations. In particular,. the depositing, migration, drying and reading operations have especially been automated and have markedly reduced the risk of errors. On the other hand, developing, at the present time, when it is necessary, is carried out either by spraying of specific reagents which allow coloured or fluorescent. spots to :be obtained, or by total immersion of the chromatographic :plate.
These techniques of developing by spraying or immersion have a certain number of dis<~dvantages.
In particular, spraying dives different results depending on the operator; gives a non-homogenous background; can require the use of compressed air, and therefore a specific apparatus; and can produce s~~reaks or drips which will render the reading difficult to interpret.
Immersion developing, fo== its part can produce a diffusion and a dilution of the spots; gives too coloured a background;
and requires a large quantity of developer.
(d) DESCRIPTION OF' THE INVEN'rION
Consequently, an object of a first aspect of the present invention is to provide an improvement in the developing step in thin layer chromatography, which better meets practical requirements than the developing procedures used hitherto, especially in that it has th~= advantage of increasing, in an unexpected manner, the repro~~ucibility and the repeatability of the thin layer chromatogr,~phy techniques, more especially in their quantitative applic,~tions.
An object of a second aspect of the present invention is to provide a developing device for carrying out that improvement.
An object of a third aspect of the present invention is to provide an apparatus containing that device.
By one broad aspect of t:zis invention, an improvement is provided in a thin-layer chromatography developing procedure in which a chromatographic plate to be developed is brought into contact with a material impregnated with a developing reagent. The improvement includes pressing, in substantially uniform manner, a porous polymer containing the developing reagent, against the thin-layer chromatography plate to be developed. The porous polymer possesses. sufficient absorbing power completely to impregnate the chromatographic plate without immersing it during the contact. under pressure, and a capacity for reabsorbing excess reagent when the porous polymer is withdrawn from the plate.
By another broad aspect of this invention, a developing device is provided for thin-layer chromatography. The device includes a porous polymer which is resistant to conventional chromatography developing reagents and which has sufficient absorbing power completely to impregnate a chromatographic plate without immersing it at the time of contact under pressure and to reabsorb excess reagent when the porous material is withdrawn from the chromatographic plate. The device also includes a manipulation support. which is inert with respect to the developing reagents, and which has sufficient rigidity to allow an appropriate pressure to be applied to the thin-layer chromatographic ~>late and on which the porous material is mounted or fixed..
By a first variant of th~~ improved procedure and the device aspects of this invention, the polymer is an open-cell polyurethane foam.
By a second variant of the improved procedure and the device aspects of this invention, and/or the above first variant thereof, the device is cylindrical in shape. By a first variation thereof, the cylindrical shape is a roll, or has a semi-cylindrical convex surface.
By a third variant of th? improved procedure and the device aspects of this invention, and/or the above first variant thereof, the device is rectangular in shape.
By a fourth variant of the improved procedure and the device aspects of this invention, and/or the above variants thereof, the device is an apparatus which further includes means for guiding and displacing at least one of the device and the chromatographic plate to be develop>ed. By a first variation of such apparatus, t=he apparatus includes an impregnation zone for impregnating the developing device and a developing zone in which the thin-layer chromatographic plate to be developed is located, and the guiding means is adapted to displace the device between a position in which it is located in the impregnation :?one and a rest zone, the displacement causing the pre;~surization of the device with the thin-layer chromatographic p:Late, and the impregnation of the plate by the developing reagf~nt.
By a fifth variant thereof and/or the above variants thereof, a dewatering zone is provided for dewatering the device, the dewatering zone being located between the impregnation zone and the developing zone.
By a sixth variant thereof, and/or the above variants thereof, the device comprises a roll which is cylindrical in shape, the roll comprising an axial shaft, the ends thereof being received in guiding slides which extend laterally on either side of the impregnation, developing and rest zones.
By a seventh variant thereof, and/or the above variants thereof, the device is in th~~ form of a box, the box including a pivotally-mounted lid, the pivotally-mounted lid allowing access to the impregnation, ~~eveloping and rest zones, delimiting the slides with fixed lateral walls of the box.
By an eighth variant thereof, and/or the above variants thereof, the device comprises a cylindrical roll which is rotatably mounted about a fixed axis, a roll of porous material impregnated with th~~ reagents rolled on the cylindrical surface of the cylindrical roll, the means for guiding the chromatographic plate being spaced from the cylindrical roll in a plane tangential to the roll, the spacing being such as to ensure a predetermined contact pressure of the porous material against the plate and to bring about impregnation of the plate by the developing reagent. By a first variation thereof, the means for guiding the chromatographic plate to be developed is formed by a second roll which is rotatable about a fixed axis that is parallel to that of the first roll and which is vertically spaced therefrom.
In particular, spraying dives different results depending on the operator; gives a non-homogenous background; can require the use of compressed air, and therefore a specific apparatus; and can produce s~~reaks or drips which will render the reading difficult to interpret.
Immersion developing, fo== its part can produce a diffusion and a dilution of the spots; gives too coloured a background;
and requires a large quantity of developer.
(d) DESCRIPTION OF' THE INVEN'rION
Consequently, an object of a first aspect of the present invention is to provide an improvement in the developing step in thin layer chromatography, which better meets practical requirements than the developing procedures used hitherto, especially in that it has th~= advantage of increasing, in an unexpected manner, the repro~~ucibility and the repeatability of the thin layer chromatogr,~phy techniques, more especially in their quantitative applic,~tions.
An object of a second aspect of the present invention is to provide a developing device for carrying out that improvement.
An object of a third aspect of the present invention is to provide an apparatus containing that device.
By one broad aspect of t:zis invention, an improvement is provided in a thin-layer chromatography developing procedure in which a chromatographic plate to be developed is brought into contact with a material impregnated with a developing reagent. The improvement includes pressing, in substantially uniform manner, a porous polymer containing the developing reagent, against the thin-layer chromatography plate to be developed. The porous polymer possesses. sufficient absorbing power completely to impregnate the chromatographic plate without immersing it during the contact. under pressure, and a capacity for reabsorbing excess reagent when the porous polymer is withdrawn from the plate.
By another broad aspect of this invention, a developing device is provided for thin-layer chromatography. The device includes a porous polymer which is resistant to conventional chromatography developing reagents and which has sufficient absorbing power completely to impregnate a chromatographic plate without immersing it at the time of contact under pressure and to reabsorb excess reagent when the porous material is withdrawn from the chromatographic plate. The device also includes a manipulation support. which is inert with respect to the developing reagents, and which has sufficient rigidity to allow an appropriate pressure to be applied to the thin-layer chromatographic ~>late and on which the porous material is mounted or fixed..
By a first variant of th~~ improved procedure and the device aspects of this invention, the polymer is an open-cell polyurethane foam.
By a second variant of the improved procedure and the device aspects of this invention, and/or the above first variant thereof, the device is cylindrical in shape. By a first variation thereof, the cylindrical shape is a roll, or has a semi-cylindrical convex surface.
By a third variant of th? improved procedure and the device aspects of this invention, and/or the above first variant thereof, the device is rectangular in shape.
By a fourth variant of the improved procedure and the device aspects of this invention, and/or the above variants thereof, the device is an apparatus which further includes means for guiding and displacing at least one of the device and the chromatographic plate to be develop>ed. By a first variation of such apparatus, t=he apparatus includes an impregnation zone for impregnating the developing device and a developing zone in which the thin-layer chromatographic plate to be developed is located, and the guiding means is adapted to displace the device between a position in which it is located in the impregnation :?one and a rest zone, the displacement causing the pre;~surization of the device with the thin-layer chromatographic p:Late, and the impregnation of the plate by the developing reagf~nt.
By a fifth variant thereof and/or the above variants thereof, a dewatering zone is provided for dewatering the device, the dewatering zone being located between the impregnation zone and the developing zone.
By a sixth variant thereof, and/or the above variants thereof, the device comprises a roll which is cylindrical in shape, the roll comprising an axial shaft, the ends thereof being received in guiding slides which extend laterally on either side of the impregnation, developing and rest zones.
By a seventh variant thereof, and/or the above variants thereof, the device is in th~~ form of a box, the box including a pivotally-mounted lid, the pivotally-mounted lid allowing access to the impregnation, ~~eveloping and rest zones, delimiting the slides with fixed lateral walls of the box.
By an eighth variant thereof, and/or the above variants thereof, the device comprises a cylindrical roll which is rotatably mounted about a fixed axis, a roll of porous material impregnated with th~~ reagents rolled on the cylindrical surface of the cylindrical roll, the means for guiding the chromatographic plate being spaced from the cylindrical roll in a plane tangential to the roll, the spacing being such as to ensure a predetermined contact pressure of the porous material against the plate and to bring about impregnation of the plate by the developing reagent. By a first variation thereof, the means for guiding the chromatographic plate to be developed is formed by a second roll which is rotatable about a fixed axis that is parallel to that of the first roll and which is vertically spaced therefrom.
By a ninth variant thereof, and/or the above variants thereoa, at least one of the two rolls is driven in rotation about its axis.
By a tenth variant of the improved 1?rocedure and the device aspects of this invention, and/o:r the above variants thereof, the apparatus further includes a zone for impregnation with reagent, tree zone being located below the first .roll.
By an eleventh variant thereof, and/or the above variants thereof, the first roll has a cylindrical surface which is divided into a plurality of compartments or sectors which are placed side by side along the axis of t:he roll.
By a twelfth variant thereof, and/o:r the above variants thereof, the means for guiding the chromatographic plate is formed by slides which are oriented in the plane perpendicularly to the axis of the device, the means for guiding thereby receiving edges of the developing plate or of its support, and the apparatus includes pulling or pushing means to displace the plate along the slides. By a variation thereof, the slides are groo~res or rails which are mounted on a support casing of the first= roll.
The term "Pressure" in the present invention is understood as meaning the action of pre;~sing or pushing with force, the pressure being capable of be-ing applied manually or mechanically.
According to an advantageous embodiment of a first aspect of this invention, the suitable material consists of a porous material which is resistant to the usual chromatography developing agents and possesses an adequate absorbing capacity completely to impregnate the chromatographic plate, without immersing it during contacting under pressure, and to reabsorb excess agent on removal. of t:ze material from the chromatographic plate. According to a still more advantageous form of this embodiment, the porous material is a suitable polymer.
By a tenth variant of the improved 1?rocedure and the device aspects of this invention, and/o:r the above variants thereof, the apparatus further includes a zone for impregnation with reagent, tree zone being located below the first .roll.
By an eleventh variant thereof, and/or the above variants thereof, the first roll has a cylindrical surface which is divided into a plurality of compartments or sectors which are placed side by side along the axis of t:he roll.
By a twelfth variant thereof, and/o:r the above variants thereof, the means for guiding the chromatographic plate is formed by slides which are oriented in the plane perpendicularly to the axis of the device, the means for guiding thereby receiving edges of the developing plate or of its support, and the apparatus includes pulling or pushing means to displace the plate along the slides. By a variation thereof, the slides are groo~res or rails which are mounted on a support casing of the first= roll.
The term "Pressure" in the present invention is understood as meaning the action of pre;~sing or pushing with force, the pressure being capable of be-ing applied manually or mechanically.
According to an advantageous embodiment of a first aspect of this invention, the suitable material consists of a porous material which is resistant to the usual chromatography developing agents and possesses an adequate absorbing capacity completely to impregnate the chromatographic plate, without immersing it during contacting under pressure, and to reabsorb excess agent on removal. of t:ze material from the chromatographic plate. According to a still more advantageous form of this embodiment, the porous material is a suitable polymer.
Such a developing procedure, called herein "Derivation Under Pressure" (DUP), has the advantage of producing a uniform background and increasing, in an unexpected manner, the reproducibility and the ~~epeatability of thin layer chromatography techniques, especially quantitative techniques.
Such a procedure also has the advantage of being easily automated.
According to an advantageous embodiment of a second aspect of this invention, the device includes a suitable polymer as porous material. The polymer advantageously possesses a foam or sponge-like structure.
According to a further advantageous feature of this embodiment of this second aspect of this invention, the polymer is a polyurethane fo<~m with open pores.
According to yet another advantageous embodiment of the device of the second aspect of this invention, the device of cylindrical shape, e.g., a roller, or one possessing a convex semi-cylindrical surface.
According to yet another advantageous embodiment of the device of the second aspect of this invention, the device is of rectangular shape.
The device necessarily possesses a flat zone of contact with the chromatographic plan=e, in order to apply a substantially-constant and uniform pressure on the chromatographic plate, regardless of the zone of contact.
In accordance with an embodiment of a third aspect of this invention, the device in thi:~ apparatus is advantageously a roller of cylindrical shape, and includes an axial shaft whose ends are accommodated in guiding channels extending laterally on either side of the impregnation, developing and rest zones.
According to another advantageous arrangement of this embodiment of the apparatus ~~f this third aspect of this invention, the apparatus is in the form of a box with a pivoting lid, which allows access to the impregnation, developing and rest zones, and delimits the channels with fixed lateral walls of the box.
Ac~~ording to another advantageous embodiment of the apparatus of this third aspect of this invention, means are provided for guiding and for moving the chromatographic plate to be developed. In such embodiment of the apparatus of this third aspect of this invention, the device includes a cylindrical roller which is mounted for rotation about a fixed axis and on whose cylindrica_L surface is wound the porous material which is impregnated with the reagents and the guiding means move the chromatographic plate, which is situated at a distance from -she cylindrical roller, which is chosen to ensure a defined c«ntact pressure of the porous material on the plate and to carry out the impregnation of the plate by the developing agent, in a plane which is tangential to the roller.
According to yet another advantageous arrangement of this embodiment of the apparatus of this third aspect of this invention, the means for gui~~ing the chromatographic plate to be developed consists of a second roller, revolving around a fixed axis, parallel to that of the first roller and vertically-separated from the latter.
According to an advantageous form of this arrangement, at least one of the two rollers is driven in rotation about its axis. The second roller has the advantage of simultaneously interacting with the first roller in the guiding of the chromatographic plate and allowing the application of a uniform and homogeneous pressure, applied by the developing device of cylindrical shape or roller to the plate to be developed.
According to still another advantageous arrangement of this embodiment of the apparatus of this third aspect of this invention, the apparatus includes an impregnation zone for impregnating with reagent, the impregnation zone being situat=ed under the first. roller.
_g_ According to yet still another advantageous arrangement of this embodiment of the apparatus of this third aspect of this invention, the cylindrical surface of the first roller is divided into several compartments or sectors, juxtaposed along the axis of the roller. The :impregnation zone can be advantageously divided into ~:ompartments in the same manner as the roller. Such an impregna-~i_on zone has the advantage of allowing the developing, on -she same plate, of various chromatograms, using 2 t:o ~~n" developers, which are situated in different compartments of the compartmentalised tank.
According to yet another arrangement of this embodiment of the apparatus of this third inspect of this invention, the means for guiding the chromatographic plate consist of channels which are oriented in the plane perpendicularly to the axis of the device, receiving the edges of the developing plate or its support, and drawing or pushing means are provided for moving the plate along the channels.
According to an advantag~=ous form of this arrangement, the channels are grooves or rails which are mounted on a support casing of the first roller mentioned.
(e) DESCRIPTION OF THE FIGURES
In the accompanying drawings, Figure 1 represents a calibration curve for the procedure in accordance with an aspect of the invention;
Figure 2 is a perspective projection of a square-shaped embodiment of the device in accordance with an aspect of the present invention;
Figure 3 represents another square-shaped embodiment of the device in accordance with an aspect: of the invention;
Figure 4 represents a cylindrical embodiment of the device in accordance with an embodiment of an aspect of the invention;
Figure 5, 6 and 7 represent an embodiment of the apparatus in accordance with an aspect of the invention in which Figures and 6 are cross-sectional views and Figure 7 is a perspective view;
Figure 8 is a perspective projection of an embodiment of an apparatus in accordance with an aspect of the invention, comprising a cylindrical developing device and whose means for guiding the chromatographic plate to be developed are represented by a second roller;
Figures 9 and 10 are cro:~s-sectional views of the apparatus in Figure 8;
Figure 11 is a perspective projection of another embodiment of an apparatus i~ accordance with an aspect of the invention, comprising a cylizdrical_ developing device and whose means for guiding the chromatographic. plate to be developed consist of a seconds roller;
- Figure 12 is a perspective projection of an embodiment of an apparatus in accordance with an aspect of the invention, comprising a cylindrical developing device and whose means for guiding the chromatographic plate to be developed consist of rails, receiving the edges of a support of the developing plate and associated with means for drawing the plate along the rails.
(f) AT LEAST ONE MODE FOR CARRYING OUT THE INVENTION
The following examples are given purely by way of illustration of the invention.
Example 1:
Thin layer chromatography for the determination of tryptophan is carried out as follows:
- plates; non-fluorescent cellulose 20 x 20 cm (available from MERCK) Mobile phase: 1-Butanol . 80 volumes Acetic arid . 20 volumes Water . 100 volumes Samples: tryptophan solution at 2 mg/m7_ Deposits: 10 deposits of 2 u1_, carried out using the equipment LINOMA'),TM IV (available from c:AMAG) Developing agent : ninhydri_n/c:opper Solution A . ninhydrin . 1.6 g ethanol . 800 ml acetic acid . 160 ml 2 , 4, 6-collidine . 32 rnl Solution B: cupric nitrate at. 2o in ethanol 100 ml of solution A and 6 ml of solution B are mixed to obtain the developing agent.
A developing device in accordance with an embodiment of an aspect of the invention is impregnated with the developing agent above. The device, impregnated under pressure, is applied to the chromatograph=is plate at the time of developing. Photodensitometr_ic reading is carried out using an apparatus known by the trade name CD60 (available from DESAGA). The plates are read before and after developing using the device in accordance with an embodiment of an aspect of the invention.
1. Undeveloped chromatoa:ram - reading at 254 nm (direct UV
DEPOSIT AREA HEIGHT
1 789.1 173.48 2 637,8 143.91 3 715.5 117.09 4 827.0 193.25 888.4 205.85 6 859.4 205.44 7 879.1 209.75 g 873.1 203.51 g 897.7 202.95 874.5 182.27 m = 824.16 m = 188.75 6 = 85.958 a = 21.619 CV = 10.43 o CV = 11.45 0 The repeatability is determined by the coefficient of variation (CV); the smaller t=he coefficient of variation, the better the repeatability.
2. Chromatogram after developing with ninhydrin using the developing device under pressure - reading at 510 nm:
DEPOSIT AREA HEIGHT
1 2312.9 460.27 2 2136.7 428.67 3 2225.0 467.70 4 2268.3 482.02 2209.7 471.81 6 2122.8 460.02 7 2142.7 465.76 8 2174.3 462.84 9 2188.1 465.28 2207.4 422.76 m = 21~a8.8 m = 456.71 a = 'i9.835 = 18.411 CV = 2.72 o CV = 4.03 0 The developing device according to an embodiment of an aspect of the invention allows, in an unexpected manner, results and a greatly increased precision relative to the method of reference (MR) to oe obtained.
Example 2 .
The results obtained in Example 1 are compared with those obtained when, under identical conditions, developing is carried out by spraying.
1. Undeveloped chromatogram - reading at 254 nm (directed UV) .
DEPOSIT AREA HEIGHT
1 922.1 181.59 2 918.1 179.51 3 869.5 175.36 4 871.4 170.30 842.5 167.04 6 833.9 164.93 7 835.3 166.51 8 864.8 171.63 9 829.6 170.97 870.8 174.08 m =- 86_'x.8 m = 172.19 o = 32.96 0 = 5.502 CV = 3.81 o CV = 3.19 0 2. Chromatogram after developing with ninhydrin by conventional spraying - reading at 510 nm .
DEPOSIT AREA HEIGHT
1 1686.9 321.07 2 1639.0 312.03 3 1729.3 331.35 4 1743.1 332.25 5 1676.3 327.02 6 1614.7 313.29 7 1508.7 290.53 8 1455.4 283.81 9 1493.4 296.73 10 1482.8 290.04 m = 1602.96 m = 309.812 6 = 10f3.81 0 = 18.309 CV = 6.79 o CV = 5.91 When the conventional developing method, by spraying, is used, the results are i_n.ferior to direct MR (Method of Reference).
Example 3 . Study of the relationship between the signal detected and the quantity deposited. 10 deposits of 4u1 of tryptophan solutions at 0.1 mg/ml, 0.2 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml were depos__ted on a thin layer chromatographic plate in cel-_ulose 20 x 20 (non-fluorescent) (available from MERCK, DARMS~,ADT, F. R. G. ) .
Readings were carried out. before developing, at 275 nm, and after developing with ninhydrin, at 288 nm. Developing is carried out on the one hand by spraying and on the other using DUP (i.e.,Derivation Under Pressure) in accordance with the procedure of an aspect of the invention.
As shown by the calibrat__on curves (Figure 1), there is a resemblance between the direct MR (Method of Reference) reading (curve 2) and DUP (Derivation Under Pressure) (curve 1) in contrast to developing by spraying (curve 3) which becomes linear.
The equations of the cur~res are as follows:
Direct MR (Method of Reference 1) at 275 nm (curve 2) . Y = 0.041 x 10 o.omax r = 0.9958 DUP (Derivation Under Pressure) at 288 nm (curve 1) . Y = 0.047 x 10°~ooz~x r = 0.9972 Spraying at 288 nm (curve 3) . Y = 0.0095 X - 8.5226 r = 0.9946 Example 4 . Detection and quantitation of water-soluble vitamins.
Test solutions containing 9 water-soluble vitamins were prepared in increasing concentrations.
A first solution was prepared as follows:
Vitamin C . 2.9 g Vitamin B1 . 24.6 mg Vitamin B2 . 32.6 mg 'Jitamin B6 . 24.2 mg Vitamin B12 . 0.9 mg Biot__n . 2.42 mg Folic acid . 4.88 mg Niac__namide . 160.6 mg Ca pantothenate: 121.2 mg Ddater: to 100 ml ThE=_ other solutions correspond to a half and to a quarter dilution of the above solution.
deposits of 10 ~1 of these solutions were applied to silica plates HPTLC (High Performance T:nin Layer Chromatography) - 60 F 254 - 20 X 20 (available from MERCK, DARMST:?~DT - F.R.G.) 2 cm frorl the edge of the plate. Migration is carried out by OPLC (Over Pressure Liquid Chromatography), according to the following conditions:
Flow rate: 0.2 ml/min developing: 16 c:m Pressure of the water pump . 25 bars Pressure of the elution pump . 4 bars Pre-run using hexane.
The plate is air-dried, reading is carried out under direct MR (Method of Reference) at 254 nm for vitamins Bl, Bz, C and niacinamide, then at 2'~0 nm for folic acid and vitamin B6.
Vitamin Blz and biotin ar~~ recorded at 530 nm after DUP
(Derivation Under Pressure) using a 0.10 (w/v) solution of 4-dimethyl-aminocinnamaldehyde and 1° (v/v) of sulphuric acid in ethanol.
Calcium pantothenate is :read at 375 nm after DUP
(Derivation Under Pressure), using ninhydrin.
Quantitation is carried out by reflection densitometric reading using the peak surfaces obtained, as shown in the following table:
Vitamins Standard Results Linearity deviation (o) (r) ( ) n = 10 Direct. MR Bl 1.40 97.6 0.9999567 detection B2 6.61 99.9 0.9999607 254 nm Niacinamide 2.23 98.8 0.99998054 C 1.23 97.8 0.999976 290 nm Folic acid 11.35 95.6 0.99995382 B6 7.56 100.0 0.9999805 DUP B12 4.82 100.8 0.99958 with cinna- Biotine 3.51 101.7 0.999256 maldehyde DUP Calcium with pantothenate 2.51 97.22 0.99915 ninhydrin Example 5 . Study of the pressure to be applied during the carrying out of the process in accordan~~e with the invention.
The thin layer chromatographic plates in cellulose 10 x 10 (non-fluorescent) (available from MERCK, DARMSTADT - F.R.G.) were developed with Dragendorff's reagent using derivation under pressure, in accordance with the method of an aspect of this invention, on a 5 x 10 cm surface. The 4.5 mm thick polymer used was compressed ==or 5 seconds using 500 g, 1000 g, 2000 g and 5000 g weights as well as developing without pressure, which corresponds ~~o simply applying the polymer to the plate.
Densitometric reading of the background noise obtained was carried out; the wavelength used was 350 nm, the absorption maximum of the Dra.gendorff's reagent. Zero was obtained from the undeveloped part of the plate.
Absorbances read:
Depositing without. pressure: 0 g/50 cm~: 1810 ~ 18.70 Depositing with pressure: 500 g/.'~0 cm2: 2175 ~ 3.4a 1000 g/50 cmz: 1625 ~ 32.30 2000 g/50 cm2: 1275 ~ 21.5a 5000 g/50 cm2: 2075 ~ 3.6%
Corresponding to the pressure appliE:d to the polymer is an aspirat=ion of the reagent when this pressure is released. This is very important during applications of 1000 g and 2000 g/50 cm2 weights and produces a return of the developer inside the polymer. This return is negligible for .'000 g/50 cm2 or more and non-existent for a pressure of 500 g/50 cm2.
The absence of pressure ~~roduces a simple diffusion of the reagent towards the plate in a very non-homogeneous manner.
The optimum development is obtained for a pressure of 5000 g/50 cm2, that is to say 40 kg for a standard 20 x 20 cm plate and a 4.5 mm thick polymer.
The procedure according t:o an aspect of the invention can be carried out using one of t:he devices described hereinafter.
The device in accordance with an embodiment of an aspect of the present invention, shown in Figure 2, comprises a rigid support 10 which is equipped with a handling (gripping) member 11, on which is mounted a po:Lymeric material 20, which is impregnated with developing agent. In this embodiment of an aspect of this invention, and in a non-restrictive manner, the device has a square shape, of sides of 200 mm, in other words of the same size as the chromatographic plates. The polymeric material has a thickness of 4.5 mm, while the rigid support has a thickness of 20 mm.
The device of this embodiment of an aspect of this invention appears in cross-sectional view as an inverted ~~T"
and is used advantageously for applying the developing agent to a chromatographic plate, as described in the above example.
After use, the device of this embodiment of an aspect of this invention is stored in suitable hermetic tanks.
Figure 3 represents another embodiment of the device in accordance with an aspect of the invention in which the rigid support section 10, which is in contact. with the polymeric support 20 which is impregnated with reagent, is slightly convex.
The device in accordance with an embodiment of~ an aspect of the present. invention, shown in Figure 4, comprises a rigid support 10' in the form of: a roller, which is equipped with an operating axle 11' around which the porous polymeric material 20', which is impregnated with developing agent is applied, as described in t:he above example.
The devices may be used in an apparat=us in accordance with an embodiment of an aspe~~t of the inventic>n, as shown in Figures 5, 6 and 7 for apL_>lying the developing agent by rolling on a c:hromatograpruic plate 30.
Figure 5 represent=. an embodiment of_ an apparatus in accordance with an aspE:ct of the invention in the form of a box 2, which includes an z.mpregnati.on zone A for the device 1, represented in this apparatus by a tank in which a developing agent is present, as described in Example 1. It also includes a developing zone C, where the chromatographic plate 30 to be developed is situated and which furthermore comprises the plate support: 311 and 31~, as well as the stops 321 and 322,, which block the plate. It also includes means 40 for guiding and moving the device 1 between a position la, where it is present in the impregnation zone, and a position 1d, called the arrest" position (zone D) . The ends c>f the axle 11' (See FIG. 4) of them device are thus accommodated in the channe l; 41 of the means 40 extending above the chromatographic plate 30.
It further includes a zone B for draining the device 1 (position 1b of the de~rice), which is situated between the impregnation :?one A anti the developing -none C.
Such an apparatus allows a constant and uniform pressure to be applied to the p:Late 30 and thereby allows a greatly--improved deve:Loping to be obtained, as shown in Examples 1 and 2 above.
A device L in accordance with an embodiment of an aspect of the invention is fi:r_st.:Ly placed in the impregnation zone A, in position la and is thus impregnated with developing agent.
Then the deviate is transferred to position 1b, in the draining zone B, in which any excess reagent which may be present is drained, before passing the device 1 intc> the developing zone C, in which the reagent is released from the device and uniformly impregnates the plate to be developed.
Figure 6 represents the apparatus, with the detachable section 50a of the pivoting lid 50, which allows access to the impregnation anal developing zones, when opened. The lid has two lateral edges which are cut into step-like shapes, the edges of the lid delimiting the channels 41 of the means 40 (See FIG. 5)wit.h the lateral walls 60 of the box 2, cut so as to correspond t.o the edges.
Figure 7 represents a perspective projection of the apparatus as described in Figures 5 and 6.
The section of the apparatus represented in Figure 8 includes a developing devic_ve in the form of a roller R1 which comprises a rigid support 70 of cylindrical. shape rotating around a fixed axis of z:otation 71 and on whose surface a band 72 of porous polymeric nuaterial, impregnated with developing agent is applied, as described in the above example. The roller R1 comprises a means for fixing the porous polymeric material 72, formed, for. example, by pincers 73 placed along a generatrix of t:he rolle~:~, and embedded in t:he latter so that it does not project above r_he cylindrical. surface of the roller. These pincers 73 a:Llow the porous material 72 to be fixed by one of its end;, with the c5ther end remaining free.
The roller R1 rotates around the fixed axis 71 in a clockwise direction in Figure 1, teeing driven in rotation by any suitable means,, especia:l_ly a mechanical means, e. g. , a Iran)':
or a motor. It also inc:Ludes a reagent tank 80, which may be removable, situated undf=r the roller R1. It. also includes a roller RZ which is rotat.abl.e around a fixed axis 91,, which is parallel to that of the ro:Ller Rl and vertically separated from the latter. This roller R.2 is driven by suitable means and especially, in a non-restrictive manner, by gear wheels, during the rotation of 'the roller R,, in the opposite direction to the latter and at identical speed. It: also includes the chromatographic plate to be developed P, as shown in Figures 2 and 3, which is sandwiched between the rollers R1 and R2. The plate is driven by the two rollers and rnoved in contact with the porous material, under a defined pressure depending on the thickness of the plate P, the thickness of the porous material and the distance separating the two rollers.
Figures 9 and :LO show cross-sectional views of the apparatus in Figure 8. The reagent tank 80, which is filled with a suitable reagent, is ~>laced under the roller R1. The porous polymeric band 72 i_s fixed inside the pincers of the roller R1. During the first rotation of the roller Rl, the porous material 72 takes the shape of the roller. On arrival inside the tank 80, it is impregnated with reagent in a continuous manner, during each turn. A rotation can be carried out to pre-impregnate the material 72, in order to saturate it with reagent, before passing off the plate P.
The plate P to be developed is placed so that its stationary phase is in contacts with the porous material 72.
The developing is carried oui= progressively during the rotation and excess reagent upstream of the developing zone is discarded into the tank 80. Lxcess reagent after developing is, for its part, reabsorbed by the porous material 72.
Figure 10 shows more spe~~ifically the free end of the band of porous material 72, and a casing X in which the roller R1 is housed and protected.
Figure 11 represents a roller R, comprising, in the example represented, 5 bands (n: to n5) of porous material 72, which are separated from one another by grooves r1 to r4, which bands are respectively impregnated in compartments 801 to 805 of the compartmentalised reagent tank 80. This; apparatus thus allows different chromatograms to be developed on the same chromatographic plate, i_n the embodiment represented, using five different developers.
Figure 12 shows an embodiment of the apparatus of an aspect of this invention in which the p:Late P, which is placed on a support guided on rails or channels 100, passes under the roller Rl, rotating around the fixed axis 77_. The space between the porous material 72 covering the roller and the plate P is defined so as to provide a sufficient pressure for the developing procedure. The movement of the movable support carrying the plate P is produced by pushing or pulling. For that purpose, any suitable means of moving the support can be used. A belt conveyor formin<~ this support can alternatively be used. The contact between the plate P and the roller R1 allows the latter to rotate <~nd the porous material 72 to be compressed, the roller R, remaining well in place during the passing of the plate P by vi:rt:ue of the closure of the cover C.
Such a procedure also has the advantage of being easily automated.
According to an advantageous embodiment of a second aspect of this invention, the device includes a suitable polymer as porous material. The polymer advantageously possesses a foam or sponge-like structure.
According to a further advantageous feature of this embodiment of this second aspect of this invention, the polymer is a polyurethane fo<~m with open pores.
According to yet another advantageous embodiment of the device of the second aspect of this invention, the device of cylindrical shape, e.g., a roller, or one possessing a convex semi-cylindrical surface.
According to yet another advantageous embodiment of the device of the second aspect of this invention, the device is of rectangular shape.
The device necessarily possesses a flat zone of contact with the chromatographic plan=e, in order to apply a substantially-constant and uniform pressure on the chromatographic plate, regardless of the zone of contact.
In accordance with an embodiment of a third aspect of this invention, the device in thi:~ apparatus is advantageously a roller of cylindrical shape, and includes an axial shaft whose ends are accommodated in guiding channels extending laterally on either side of the impregnation, developing and rest zones.
According to another advantageous arrangement of this embodiment of the apparatus ~~f this third aspect of this invention, the apparatus is in the form of a box with a pivoting lid, which allows access to the impregnation, developing and rest zones, and delimits the channels with fixed lateral walls of the box.
Ac~~ording to another advantageous embodiment of the apparatus of this third aspect of this invention, means are provided for guiding and for moving the chromatographic plate to be developed. In such embodiment of the apparatus of this third aspect of this invention, the device includes a cylindrical roller which is mounted for rotation about a fixed axis and on whose cylindrica_L surface is wound the porous material which is impregnated with the reagents and the guiding means move the chromatographic plate, which is situated at a distance from -she cylindrical roller, which is chosen to ensure a defined c«ntact pressure of the porous material on the plate and to carry out the impregnation of the plate by the developing agent, in a plane which is tangential to the roller.
According to yet another advantageous arrangement of this embodiment of the apparatus of this third aspect of this invention, the means for gui~~ing the chromatographic plate to be developed consists of a second roller, revolving around a fixed axis, parallel to that of the first roller and vertically-separated from the latter.
According to an advantageous form of this arrangement, at least one of the two rollers is driven in rotation about its axis. The second roller has the advantage of simultaneously interacting with the first roller in the guiding of the chromatographic plate and allowing the application of a uniform and homogeneous pressure, applied by the developing device of cylindrical shape or roller to the plate to be developed.
According to still another advantageous arrangement of this embodiment of the apparatus of this third aspect of this invention, the apparatus includes an impregnation zone for impregnating with reagent, the impregnation zone being situat=ed under the first. roller.
_g_ According to yet still another advantageous arrangement of this embodiment of the apparatus of this third aspect of this invention, the cylindrical surface of the first roller is divided into several compartments or sectors, juxtaposed along the axis of the roller. The :impregnation zone can be advantageously divided into ~:ompartments in the same manner as the roller. Such an impregna-~i_on zone has the advantage of allowing the developing, on -she same plate, of various chromatograms, using 2 t:o ~~n" developers, which are situated in different compartments of the compartmentalised tank.
According to yet another arrangement of this embodiment of the apparatus of this third inspect of this invention, the means for guiding the chromatographic plate consist of channels which are oriented in the plane perpendicularly to the axis of the device, receiving the edges of the developing plate or its support, and drawing or pushing means are provided for moving the plate along the channels.
According to an advantag~=ous form of this arrangement, the channels are grooves or rails which are mounted on a support casing of the first roller mentioned.
(e) DESCRIPTION OF THE FIGURES
In the accompanying drawings, Figure 1 represents a calibration curve for the procedure in accordance with an aspect of the invention;
Figure 2 is a perspective projection of a square-shaped embodiment of the device in accordance with an aspect of the present invention;
Figure 3 represents another square-shaped embodiment of the device in accordance with an aspect: of the invention;
Figure 4 represents a cylindrical embodiment of the device in accordance with an embodiment of an aspect of the invention;
Figure 5, 6 and 7 represent an embodiment of the apparatus in accordance with an aspect of the invention in which Figures and 6 are cross-sectional views and Figure 7 is a perspective view;
Figure 8 is a perspective projection of an embodiment of an apparatus in accordance with an aspect of the invention, comprising a cylindrical developing device and whose means for guiding the chromatographic plate to be developed are represented by a second roller;
Figures 9 and 10 are cro:~s-sectional views of the apparatus in Figure 8;
Figure 11 is a perspective projection of another embodiment of an apparatus i~ accordance with an aspect of the invention, comprising a cylizdrical_ developing device and whose means for guiding the chromatographic. plate to be developed consist of a seconds roller;
- Figure 12 is a perspective projection of an embodiment of an apparatus in accordance with an aspect of the invention, comprising a cylindrical developing device and whose means for guiding the chromatographic plate to be developed consist of rails, receiving the edges of a support of the developing plate and associated with means for drawing the plate along the rails.
(f) AT LEAST ONE MODE FOR CARRYING OUT THE INVENTION
The following examples are given purely by way of illustration of the invention.
Example 1:
Thin layer chromatography for the determination of tryptophan is carried out as follows:
- plates; non-fluorescent cellulose 20 x 20 cm (available from MERCK) Mobile phase: 1-Butanol . 80 volumes Acetic arid . 20 volumes Water . 100 volumes Samples: tryptophan solution at 2 mg/m7_ Deposits: 10 deposits of 2 u1_, carried out using the equipment LINOMA'),TM IV (available from c:AMAG) Developing agent : ninhydri_n/c:opper Solution A . ninhydrin . 1.6 g ethanol . 800 ml acetic acid . 160 ml 2 , 4, 6-collidine . 32 rnl Solution B: cupric nitrate at. 2o in ethanol 100 ml of solution A and 6 ml of solution B are mixed to obtain the developing agent.
A developing device in accordance with an embodiment of an aspect of the invention is impregnated with the developing agent above. The device, impregnated under pressure, is applied to the chromatograph=is plate at the time of developing. Photodensitometr_ic reading is carried out using an apparatus known by the trade name CD60 (available from DESAGA). The plates are read before and after developing using the device in accordance with an embodiment of an aspect of the invention.
1. Undeveloped chromatoa:ram - reading at 254 nm (direct UV
DEPOSIT AREA HEIGHT
1 789.1 173.48 2 637,8 143.91 3 715.5 117.09 4 827.0 193.25 888.4 205.85 6 859.4 205.44 7 879.1 209.75 g 873.1 203.51 g 897.7 202.95 874.5 182.27 m = 824.16 m = 188.75 6 = 85.958 a = 21.619 CV = 10.43 o CV = 11.45 0 The repeatability is determined by the coefficient of variation (CV); the smaller t=he coefficient of variation, the better the repeatability.
2. Chromatogram after developing with ninhydrin using the developing device under pressure - reading at 510 nm:
DEPOSIT AREA HEIGHT
1 2312.9 460.27 2 2136.7 428.67 3 2225.0 467.70 4 2268.3 482.02 2209.7 471.81 6 2122.8 460.02 7 2142.7 465.76 8 2174.3 462.84 9 2188.1 465.28 2207.4 422.76 m = 21~a8.8 m = 456.71 a = 'i9.835 = 18.411 CV = 2.72 o CV = 4.03 0 The developing device according to an embodiment of an aspect of the invention allows, in an unexpected manner, results and a greatly increased precision relative to the method of reference (MR) to oe obtained.
Example 2 .
The results obtained in Example 1 are compared with those obtained when, under identical conditions, developing is carried out by spraying.
1. Undeveloped chromatogram - reading at 254 nm (directed UV) .
DEPOSIT AREA HEIGHT
1 922.1 181.59 2 918.1 179.51 3 869.5 175.36 4 871.4 170.30 842.5 167.04 6 833.9 164.93 7 835.3 166.51 8 864.8 171.63 9 829.6 170.97 870.8 174.08 m =- 86_'x.8 m = 172.19 o = 32.96 0 = 5.502 CV = 3.81 o CV = 3.19 0 2. Chromatogram after developing with ninhydrin by conventional spraying - reading at 510 nm .
DEPOSIT AREA HEIGHT
1 1686.9 321.07 2 1639.0 312.03 3 1729.3 331.35 4 1743.1 332.25 5 1676.3 327.02 6 1614.7 313.29 7 1508.7 290.53 8 1455.4 283.81 9 1493.4 296.73 10 1482.8 290.04 m = 1602.96 m = 309.812 6 = 10f3.81 0 = 18.309 CV = 6.79 o CV = 5.91 When the conventional developing method, by spraying, is used, the results are i_n.ferior to direct MR (Method of Reference).
Example 3 . Study of the relationship between the signal detected and the quantity deposited. 10 deposits of 4u1 of tryptophan solutions at 0.1 mg/ml, 0.2 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml were depos__ted on a thin layer chromatographic plate in cel-_ulose 20 x 20 (non-fluorescent) (available from MERCK, DARMS~,ADT, F. R. G. ) .
Readings were carried out. before developing, at 275 nm, and after developing with ninhydrin, at 288 nm. Developing is carried out on the one hand by spraying and on the other using DUP (i.e.,Derivation Under Pressure) in accordance with the procedure of an aspect of the invention.
As shown by the calibrat__on curves (Figure 1), there is a resemblance between the direct MR (Method of Reference) reading (curve 2) and DUP (Derivation Under Pressure) (curve 1) in contrast to developing by spraying (curve 3) which becomes linear.
The equations of the cur~res are as follows:
Direct MR (Method of Reference 1) at 275 nm (curve 2) . Y = 0.041 x 10 o.omax r = 0.9958 DUP (Derivation Under Pressure) at 288 nm (curve 1) . Y = 0.047 x 10°~ooz~x r = 0.9972 Spraying at 288 nm (curve 3) . Y = 0.0095 X - 8.5226 r = 0.9946 Example 4 . Detection and quantitation of water-soluble vitamins.
Test solutions containing 9 water-soluble vitamins were prepared in increasing concentrations.
A first solution was prepared as follows:
Vitamin C . 2.9 g Vitamin B1 . 24.6 mg Vitamin B2 . 32.6 mg 'Jitamin B6 . 24.2 mg Vitamin B12 . 0.9 mg Biot__n . 2.42 mg Folic acid . 4.88 mg Niac__namide . 160.6 mg Ca pantothenate: 121.2 mg Ddater: to 100 ml ThE=_ other solutions correspond to a half and to a quarter dilution of the above solution.
deposits of 10 ~1 of these solutions were applied to silica plates HPTLC (High Performance T:nin Layer Chromatography) - 60 F 254 - 20 X 20 (available from MERCK, DARMST:?~DT - F.R.G.) 2 cm frorl the edge of the plate. Migration is carried out by OPLC (Over Pressure Liquid Chromatography), according to the following conditions:
Flow rate: 0.2 ml/min developing: 16 c:m Pressure of the water pump . 25 bars Pressure of the elution pump . 4 bars Pre-run using hexane.
The plate is air-dried, reading is carried out under direct MR (Method of Reference) at 254 nm for vitamins Bl, Bz, C and niacinamide, then at 2'~0 nm for folic acid and vitamin B6.
Vitamin Blz and biotin ar~~ recorded at 530 nm after DUP
(Derivation Under Pressure) using a 0.10 (w/v) solution of 4-dimethyl-aminocinnamaldehyde and 1° (v/v) of sulphuric acid in ethanol.
Calcium pantothenate is :read at 375 nm after DUP
(Derivation Under Pressure), using ninhydrin.
Quantitation is carried out by reflection densitometric reading using the peak surfaces obtained, as shown in the following table:
Vitamins Standard Results Linearity deviation (o) (r) ( ) n = 10 Direct. MR Bl 1.40 97.6 0.9999567 detection B2 6.61 99.9 0.9999607 254 nm Niacinamide 2.23 98.8 0.99998054 C 1.23 97.8 0.999976 290 nm Folic acid 11.35 95.6 0.99995382 B6 7.56 100.0 0.9999805 DUP B12 4.82 100.8 0.99958 with cinna- Biotine 3.51 101.7 0.999256 maldehyde DUP Calcium with pantothenate 2.51 97.22 0.99915 ninhydrin Example 5 . Study of the pressure to be applied during the carrying out of the process in accordan~~e with the invention.
The thin layer chromatographic plates in cellulose 10 x 10 (non-fluorescent) (available from MERCK, DARMSTADT - F.R.G.) were developed with Dragendorff's reagent using derivation under pressure, in accordance with the method of an aspect of this invention, on a 5 x 10 cm surface. The 4.5 mm thick polymer used was compressed ==or 5 seconds using 500 g, 1000 g, 2000 g and 5000 g weights as well as developing without pressure, which corresponds ~~o simply applying the polymer to the plate.
Densitometric reading of the background noise obtained was carried out; the wavelength used was 350 nm, the absorption maximum of the Dra.gendorff's reagent. Zero was obtained from the undeveloped part of the plate.
Absorbances read:
Depositing without. pressure: 0 g/50 cm~: 1810 ~ 18.70 Depositing with pressure: 500 g/.'~0 cm2: 2175 ~ 3.4a 1000 g/50 cmz: 1625 ~ 32.30 2000 g/50 cm2: 1275 ~ 21.5a 5000 g/50 cm2: 2075 ~ 3.6%
Corresponding to the pressure appliE:d to the polymer is an aspirat=ion of the reagent when this pressure is released. This is very important during applications of 1000 g and 2000 g/50 cm2 weights and produces a return of the developer inside the polymer. This return is negligible for .'000 g/50 cm2 or more and non-existent for a pressure of 500 g/50 cm2.
The absence of pressure ~~roduces a simple diffusion of the reagent towards the plate in a very non-homogeneous manner.
The optimum development is obtained for a pressure of 5000 g/50 cm2, that is to say 40 kg for a standard 20 x 20 cm plate and a 4.5 mm thick polymer.
The procedure according t:o an aspect of the invention can be carried out using one of t:he devices described hereinafter.
The device in accordance with an embodiment of an aspect of the present invention, shown in Figure 2, comprises a rigid support 10 which is equipped with a handling (gripping) member 11, on which is mounted a po:Lymeric material 20, which is impregnated with developing agent. In this embodiment of an aspect of this invention, and in a non-restrictive manner, the device has a square shape, of sides of 200 mm, in other words of the same size as the chromatographic plates. The polymeric material has a thickness of 4.5 mm, while the rigid support has a thickness of 20 mm.
The device of this embodiment of an aspect of this invention appears in cross-sectional view as an inverted ~~T"
and is used advantageously for applying the developing agent to a chromatographic plate, as described in the above example.
After use, the device of this embodiment of an aspect of this invention is stored in suitable hermetic tanks.
Figure 3 represents another embodiment of the device in accordance with an aspect of the invention in which the rigid support section 10, which is in contact. with the polymeric support 20 which is impregnated with reagent, is slightly convex.
The device in accordance with an embodiment of~ an aspect of the present. invention, shown in Figure 4, comprises a rigid support 10' in the form of: a roller, which is equipped with an operating axle 11' around which the porous polymeric material 20', which is impregnated with developing agent is applied, as described in t:he above example.
The devices may be used in an apparat=us in accordance with an embodiment of an aspe~~t of the inventic>n, as shown in Figures 5, 6 and 7 for apL_>lying the developing agent by rolling on a c:hromatograpruic plate 30.
Figure 5 represent=. an embodiment of_ an apparatus in accordance with an aspE:ct of the invention in the form of a box 2, which includes an z.mpregnati.on zone A for the device 1, represented in this apparatus by a tank in which a developing agent is present, as described in Example 1. It also includes a developing zone C, where the chromatographic plate 30 to be developed is situated and which furthermore comprises the plate support: 311 and 31~, as well as the stops 321 and 322,, which block the plate. It also includes means 40 for guiding and moving the device 1 between a position la, where it is present in the impregnation zone, and a position 1d, called the arrest" position (zone D) . The ends c>f the axle 11' (See FIG. 4) of them device are thus accommodated in the channe l; 41 of the means 40 extending above the chromatographic plate 30.
It further includes a zone B for draining the device 1 (position 1b of the de~rice), which is situated between the impregnation :?one A anti the developing -none C.
Such an apparatus allows a constant and uniform pressure to be applied to the p:Late 30 and thereby allows a greatly--improved deve:Loping to be obtained, as shown in Examples 1 and 2 above.
A device L in accordance with an embodiment of an aspect of the invention is fi:r_st.:Ly placed in the impregnation zone A, in position la and is thus impregnated with developing agent.
Then the deviate is transferred to position 1b, in the draining zone B, in which any excess reagent which may be present is drained, before passing the device 1 intc> the developing zone C, in which the reagent is released from the device and uniformly impregnates the plate to be developed.
Figure 6 represents the apparatus, with the detachable section 50a of the pivoting lid 50, which allows access to the impregnation anal developing zones, when opened. The lid has two lateral edges which are cut into step-like shapes, the edges of the lid delimiting the channels 41 of the means 40 (See FIG. 5)wit.h the lateral walls 60 of the box 2, cut so as to correspond t.o the edges.
Figure 7 represents a perspective projection of the apparatus as described in Figures 5 and 6.
The section of the apparatus represented in Figure 8 includes a developing devic_ve in the form of a roller R1 which comprises a rigid support 70 of cylindrical. shape rotating around a fixed axis of z:otation 71 and on whose surface a band 72 of porous polymeric nuaterial, impregnated with developing agent is applied, as described in the above example. The roller R1 comprises a means for fixing the porous polymeric material 72, formed, for. example, by pincers 73 placed along a generatrix of t:he rolle~:~, and embedded in t:he latter so that it does not project above r_he cylindrical. surface of the roller. These pincers 73 a:Llow the porous material 72 to be fixed by one of its end;, with the c5ther end remaining free.
The roller R1 rotates around the fixed axis 71 in a clockwise direction in Figure 1, teeing driven in rotation by any suitable means,, especia:l_ly a mechanical means, e. g. , a Iran)':
or a motor. It also inc:Ludes a reagent tank 80, which may be removable, situated undf=r the roller R1. It. also includes a roller RZ which is rotat.abl.e around a fixed axis 91,, which is parallel to that of the ro:Ller Rl and vertically separated from the latter. This roller R.2 is driven by suitable means and especially, in a non-restrictive manner, by gear wheels, during the rotation of 'the roller R,, in the opposite direction to the latter and at identical speed. It: also includes the chromatographic plate to be developed P, as shown in Figures 2 and 3, which is sandwiched between the rollers R1 and R2. The plate is driven by the two rollers and rnoved in contact with the porous material, under a defined pressure depending on the thickness of the plate P, the thickness of the porous material and the distance separating the two rollers.
Figures 9 and :LO show cross-sectional views of the apparatus in Figure 8. The reagent tank 80, which is filled with a suitable reagent, is ~>laced under the roller R1. The porous polymeric band 72 i_s fixed inside the pincers of the roller R1. During the first rotation of the roller Rl, the porous material 72 takes the shape of the roller. On arrival inside the tank 80, it is impregnated with reagent in a continuous manner, during each turn. A rotation can be carried out to pre-impregnate the material 72, in order to saturate it with reagent, before passing off the plate P.
The plate P to be developed is placed so that its stationary phase is in contacts with the porous material 72.
The developing is carried oui= progressively during the rotation and excess reagent upstream of the developing zone is discarded into the tank 80. Lxcess reagent after developing is, for its part, reabsorbed by the porous material 72.
Figure 10 shows more spe~~ifically the free end of the band of porous material 72, and a casing X in which the roller R1 is housed and protected.
Figure 11 represents a roller R, comprising, in the example represented, 5 bands (n: to n5) of porous material 72, which are separated from one another by grooves r1 to r4, which bands are respectively impregnated in compartments 801 to 805 of the compartmentalised reagent tank 80. This; apparatus thus allows different chromatograms to be developed on the same chromatographic plate, i_n the embodiment represented, using five different developers.
Figure 12 shows an embodiment of the apparatus of an aspect of this invention in which the p:Late P, which is placed on a support guided on rails or channels 100, passes under the roller Rl, rotating around the fixed axis 77_. The space between the porous material 72 covering the roller and the plate P is defined so as to provide a sufficient pressure for the developing procedure. The movement of the movable support carrying the plate P is produced by pushing or pulling. For that purpose, any suitable means of moving the support can be used. A belt conveyor formin<~ this support can alternatively be used. The contact between the plate P and the roller R1 allows the latter to rotate <~nd the porous material 72 to be compressed, the roller R, remaining well in place during the passing of the plate P by vi:rt:ue of the closure of the cover C.
Claims (18)
1. In a thin-layer chromatography developing procedure in which a chromatographic plate to be developed is brought into contact with a material impregnated with a developing reagent, the improvement which comprises:
pressing, in substantially uniform manner, a porous polymer containing said developing reagent, against said thin-layer chromatography plate to be developed; and wherein said porous polymer possesses (i) sufficient absorbing power completely to impregnate said chromatographic plate without immersing it during the contact under pressure, and (ii) a capacity for reabsorbing excess reagent when said porous polymer is withdrawn from said plate.
pressing, in substantially uniform manner, a porous polymer containing said developing reagent, against said thin-layer chromatography plate to be developed; and wherein said porous polymer possesses (i) sufficient absorbing power completely to impregnate said chromatographic plate without immersing it during the contact under pressure, and (ii) a capacity for reabsorbing excess reagent when said porous polymer is withdrawn from said plate.
2. Developing device for thin-layer chromatography, comprising:
(a) a porous polymer which is resistant to conventional chromatography developing reagents and which has sufficient absorbing power completely to impregnate a chromatographic plate without immersing it at the time of contact under pressure and to reabsorb excess reagent when said porous material is withdrawn from said chromatographic plate; and (b) a manipulation support which is inert with respect to said developing reagents, and which has sufficient rigidity to allow an appropriate pressure to be applied to said thin-layer chromatographic plate end on which said porous material is mounted or fixed.
(a) a porous polymer which is resistant to conventional chromatography developing reagents and which has sufficient absorbing power completely to impregnate a chromatographic plate without immersing it at the time of contact under pressure and to reabsorb excess reagent when said porous material is withdrawn from said chromatographic plate; and (b) a manipulation support which is inert with respect to said developing reagents, and which has sufficient rigidity to allow an appropriate pressure to be applied to said thin-layer chromatographic plate end on which said porous material is mounted or fixed.
3. Device according to Claim 2, wherein said polymer is an open-cell polyurethane foam.
4. Device according to Claim 2 or Claim 3, wherein said device is cylindrical in shape.
5. Device according to Claim 4 wherein said cylindrical shape is a roll, or has a semi-cylindrical convex surface.
6. Device according to Claim 2 or Claim 3, wherein said device is rectangular in shape.
7. Apparatus according to claims 2 to 6, and further including:
(c) means for guiding and displacing at least one of said device and said chromatographic plate to be developed.
(c) means for guiding and displacing at least one of said device and said chromatographic plate to be developed.
8. Apparatus according to claim 7 and further including:
(d) an impregnation zone for impregnating said developing device and a developing zone in which said thin-layer chromatographic plate to be developed is located; and (e) wherein said guiding means is adapted to displace said device between a position in which it is located in the impregnation zone and a rest zone;
said displacement causing the pressurization of said device with said thin-layer chromatographic plate, and the impregnation of said plate by said developing reagent.
(d) an impregnation zone for impregnating said developing device and a developing zone in which said thin-layer chromatographic plate to be developed is located; and (e) wherein said guiding means is adapted to displace said device between a position in which it is located in the impregnation zone and a rest zone;
said displacement causing the pressurization of said device with said thin-layer chromatographic plate, and the impregnation of said plate by said developing reagent.
9. Apparatus according to Claim 7 or Claim 8 including:
(f) a dewatering zone far dewatering said device, said dewatering zone (f) being located between said impregnation zone and said developing zone.
(f) a dewatering zone far dewatering said device, said dewatering zone (f) being located between said impregnation zone and said developing zone.
10. Apparatus according to Claim 8 or Claim 9, wherein said device comprises a roll which is cylindrical in shape, said roll comprising an axial shaft, the ends thereof being received in guiding slides which extend laterally on either side of said impregnation, developing and rest zones.
11. Apparatus according to Claim 8, or Claim 9, wherein said device is in the form of a box, said box including:
(g) a pivotally-mounted lid, said pivotally-mounted lid allowing access to said impregnation, developing and rest zones, delimiting said slides with fixed lateral walls of said box.
(g) a pivotally-mounted lid, said pivotally-mounted lid allowing access to said impregnation, developing and rest zones, delimiting said slides with fixed lateral walls of said box.
12. Apparatus according to Claim 7, wherein said device comprises:
(h) a cylindrical roll which is rotatably mounted about a fixed axis;
(i) a roll of porous material impregnated with said reagents rolled on the cylindrical surface of said cylindrical roll;
(j) said means for guiding said chromatographic plate being spaced from said cylindrical roll in a plane tangential to said roll, said spacing being such as to ensure a predetermined contact pressure of said porous material against said plate and to bring about impregnation of said plate by said developing reagent.
(h) a cylindrical roll which is rotatably mounted about a fixed axis;
(i) a roll of porous material impregnated with said reagents rolled on the cylindrical surface of said cylindrical roll;
(j) said means for guiding said chromatographic plate being spaced from said cylindrical roll in a plane tangential to said roll, said spacing being such as to ensure a predetermined contact pressure of said porous material against said plate and to bring about impregnation of said plate by said developing reagent.
13. Apparatus according to Claim 12, wherein said means for guiding said chromatographic plate to be developed is formed by a second roll which is rotatable about a fixed axis that is parallel to that of said first roll and which is vertically spaced therefrom.
14. Apparatus according to Claim 13, wherein at least one of said two rolls is driven in rotation about its axis.
15. Apparatus according to Claim 12, Claim 13 or Claim 14 further including:
(k) a zone for impregnation with reagent, said zone being located below said first roll.
(k) a zone for impregnation with reagent, said zone being located below said first roll.
16. Apparatus according to Claims 12 to 15, wherein said first roll has a cylindrical surface which is divided into a plurality of compartments or sectors which are placed side by side along the axis of said roll.
17. Apparatus according to Claims 12 to 16, wherein said means for guiding said chromatographic plate is formed by slides which are oriented in said plane perpendicularly to the axis of said device, said means for guiding thereby receiving edges of said developing plate or of its support; and including pulling or pushing means to displace said plate along said slides.
18. Apparatus according to Claim 17, wherein said slides are grooves or rails which are mounted on a support casing of said first roll.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR89/09527 | 1989-07-13 | ||
| FR8909527A FR2649796B1 (en) | 1989-07-13 | 1989-07-13 | IMPROVEMENT FOR THE DEVELOPMENT OF THIN-LAYER CHROMATOGRAPHY, DEVELOPMENT DEVICE AND INSTALLATION COMPRISING SAID DEVICE |
| FR9001735A FR2658299B2 (en) | 1989-07-13 | 1990-02-14 | INSTALLATION COMPRISING A REVELATION DEVICE FOR THIN LAYER CHROMATOGRAPHY. |
| FR90/01735 | 1990-02-14 | ||
| PCT/FR1990/000520 WO1991001000A1 (en) | 1989-07-13 | 1990-07-11 | Device and installation of thin film chromatography, with improved development |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2035025A1 CA2035025A1 (en) | 1991-01-14 |
| CA2035025C true CA2035025C (en) | 2002-06-25 |
Family
ID=26227477
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002035025A Expired - Lifetime CA2035025C (en) | 1989-07-13 | 1990-07-11 | Device and installation of thin film chromatography, with improved development |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0435990B1 (en) |
| JP (1) | JP2921980B2 (en) |
| CA (1) | CA2035025C (en) |
| DE (1) | DE69008328T2 (en) |
| ES (1) | ES2055916T3 (en) |
| FR (1) | FR2658299B2 (en) |
| RU (1) | RU2073863C1 (en) |
| WO (1) | WO1991001000A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CZ305107B6 (en) * | 2010-11-24 | 2015-05-06 | Technická univerzita v Liberci | Chromatographic substrate for thin-layer chromatography or for column chromatography |
| CN103932679B (en) * | 2014-04-09 | 2017-01-25 | 天津大学 | Image correcting method for thin layer chromatography imaging system |
| CN104792919B (en) * | 2015-05-19 | 2016-06-15 | 齐齐哈尔医学院 | Constant current thin layer chromatograph |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3667917A (en) * | 1970-11-27 | 1972-06-06 | Baker Chem Co J T | Chromatography apparatus and method |
-
1990
- 1990-02-14 FR FR9001735A patent/FR2658299B2/en not_active Expired - Lifetime
- 1990-07-11 EP EP90910753A patent/EP0435990B1/en not_active Expired - Lifetime
- 1990-07-11 ES ES90910753T patent/ES2055916T3/en not_active Expired - Lifetime
- 1990-07-11 JP JP2510312A patent/JP2921980B2/en not_active Expired - Fee Related
- 1990-07-11 CA CA002035025A patent/CA2035025C/en not_active Expired - Lifetime
- 1990-07-11 RU SU904895004A patent/RU2073863C1/en active
- 1990-07-11 WO PCT/FR1990/000520 patent/WO1991001000A1/en active IP Right Grant
- 1990-07-11 DE DE69008328T patent/DE69008328T2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CA2035025A1 (en) | 1991-01-14 |
| RU2073863C1 (en) | 1997-02-20 |
| JP2921980B2 (en) | 1999-07-19 |
| WO1991001000A1 (en) | 1991-01-24 |
| FR2658299A2 (en) | 1991-08-16 |
| EP0435990A1 (en) | 1991-07-10 |
| FR2658299B2 (en) | 1993-05-21 |
| DE69008328T2 (en) | 1994-10-27 |
| JPH04501917A (en) | 1992-04-02 |
| DE69008328D1 (en) | 1994-05-26 |
| ES2055916T3 (en) | 1994-09-01 |
| EP0435990B1 (en) | 1994-04-20 |
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| EEER | Examination request | ||
| MKLA | Lapsed | ||
| MKEC | Expiry (correction) |
Effective date: 20121202 |