CA2083939A1 - Thin-layer chromatography process and device bringing into play a reduced pressure as the forced flow means - Google Patents
Thin-layer chromatography process and device bringing into play a reduced pressure as the forced flow meansInfo
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- CA2083939A1 CA2083939A1 CA002083939A CA2083939A CA2083939A1 CA 2083939 A1 CA2083939 A1 CA 2083939A1 CA 002083939 A CA002083939 A CA 002083939A CA 2083939 A CA2083939 A CA 2083939A CA 2083939 A1 CA2083939 A1 CA 2083939A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/94—Development
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Abstract
A thin-layer chromatography process and device bringing into play a reduced pressure as the forced flow is described where the said forced flow development device is characterized in that it comprises a development chamber (10, 11, 12), associated with the reduced pressure means for said chamber (14). The latter contains at least one chromatography plate (20) coated with a suitable adsorbant layer (21) and means (30) for hermetically covering said plate(s).
Description
~ r~ 3 PROCESS AND DEVICE FOR THIN-LAYER CHROMATOGRAPHY USING A
RED~CED PRESS~RE AS TH~ FORCED FLO~ MEANS.
The present invention relates to a thin-layer chromatography device using a reduced pressure as a forced flow.
Thin-layer chromatography (TLC), also called planar chromatography, is a method of separation by differential migration between a stationary phase and a mobile phase.
The first TLCs were carried out on silica plates who~e particle size was of the order of 15 ~,m and which were approxLmately 1 mm thick.
The separation of the chromatographed products can be improved when the diameter of the particles and/or the thickness of the plate is reduced; so-called EPTLC
(High Pefformance Thin Layer Chromatography) plates are then obtained. This improvement leads to a major disadvantage, namely an increase in the time necessary to reach a height equivalent to a given theoretical plateau, due to the reduction in the diameter of the particles.
To overcome this disadvantage, several solutions have been proposed:
a. to force the eluent and to increase its velocity across the plate by using pumps.
This solution has the following disadvantages in particular:
the adsorbant on the plates is not covered hermetically by the membrane and this leads to a limita-tion on the migration of the solvent;
the solvent can migrate above the layer of adsorbant, that is to say between the membrane and the adsorbant, and not inside the adsorbant.
To o~ercome the disadvantages of this method and e~pecially the limited migration distances, a Hungarian team developed a new method of liquid thin-layer chroma-tography, called OPTLC ( Overpressured Thin Layer Chroma-tograph~), described especially in the following article~:
E. Tyhak et al. (J. Chromat., 1979, 174, 75-81;
J. Chromat., l9al, 211, 45-51; J. Chromat., 1989, 471, . i ~ , :, , ~ 3 375-387); H.F. Hauck et al., (J. Chromat., 1983, 262, 113-120); E. Tyhak (J. Pharm. Biomed. Anal., 1987, 5, 3, 191-203); z. Witkiewicz et al. (J. Chromat., 1986, 373, 111-140) and the following French Patent Applications:
No. 2,483,248, No. 2,495,779, No. 2,575,810 and No.
RED~CED PRESS~RE AS TH~ FORCED FLO~ MEANS.
The present invention relates to a thin-layer chromatography device using a reduced pressure as a forced flow.
Thin-layer chromatography (TLC), also called planar chromatography, is a method of separation by differential migration between a stationary phase and a mobile phase.
The first TLCs were carried out on silica plates who~e particle size was of the order of 15 ~,m and which were approxLmately 1 mm thick.
The separation of the chromatographed products can be improved when the diameter of the particles and/or the thickness of the plate is reduced; so-called EPTLC
(High Pefformance Thin Layer Chromatography) plates are then obtained. This improvement leads to a major disadvantage, namely an increase in the time necessary to reach a height equivalent to a given theoretical plateau, due to the reduction in the diameter of the particles.
To overcome this disadvantage, several solutions have been proposed:
a. to force the eluent and to increase its velocity across the plate by using pumps.
This solution has the following disadvantages in particular:
the adsorbant on the plates is not covered hermetically by the membrane and this leads to a limita-tion on the migration of the solvent;
the solvent can migrate above the layer of adsorbant, that is to say between the membrane and the adsorbant, and not inside the adsorbant.
To o~ercome the disadvantages of this method and e~pecially the limited migration distances, a Hungarian team developed a new method of liquid thin-layer chroma-tography, called OPTLC ( Overpressured Thin Layer Chroma-tograph~), described especially in the following article~:
E. Tyhak et al. (J. Chromat., 1979, 174, 75-81;
J. Chromat., l9al, 211, 45-51; J. Chromat., 1989, 471, . i ~ , :, , ~ 3 375-387); H.F. Hauck et al., (J. Chromat., 1983, 262, 113-120); E. Tyhak (J. Pharm. Biomed. Anal., 1987, 5, 3, 191-203); z. Witkiewicz et al. (J. Chromat., 1986, 373, 111-140) and the following French Patent Applications:
No. 2,483,248, No. 2,495,779, No. 2,575,810 and No.
2,580,406. The main feature of this method lies in the fact that the layer of adsorbant of the plate is completely covered with a flexible membrane which is pressurised and the mobile phase is introduced into the thin layer, under pressure, using a pump.
~ device for implementing this method is desc-ribed especially as follows in Patent Application 2,483,248 or Patent Application 2,580,406: a TLC plate placed on a glass or plastic support, comprising a lower part and an upper part, is completely covered on its upper part with a plastic membrane (pressure membrane~, which tightly fits its surface due to the gas pressure applied in the space delimited by the membrane and the upper part of the support unit of the device.
The gas inlet a~d the pressure gauge (manometer, for example), in combination with a syringe for intro-ducing the solvent and an inlet for introducing the sample under pressure, are in the upper part of the support unit. In this device, the external pressure on the mem~rane must always be greater than the pressure exerted on the eluent (solventJ and thus leads to an overpressure. The upper part and the lower part of the support unit are held together by means of clips.
~his device and the process which follows from it have a certain number of advantages and especially make it possi~le to optimise the separation conditions by adjusting the eluent flow rate. ~owever, OPTLC never-theless has a certain number of disadvantages:
- the necessity to prepare the chromatography plates, prior to their use, to create 4 sealed joins;
- the presence of a vapour phase in the adsorbent layer during development; indeed, such a vapour phase has the disadvantage o~ altering the selectivity (separation power) of the mobile phase, especially in the case of mixtures of solvents, of reducing the distance covered by the solutes (R~) from 20 to 40%, of reducing the maximum R~, beyond which no separation is possible, to values of between 0.6 and 0.8 (formation of a virtual front) and also of leading to demixing of the mobile phase and modification of the composition of the latter;
- the necessity especially for two pressure pumps, an eluent pump and a pump for pressurising the plate via the pressure membrane, which adheres hermeti-cally to the said plate.
- Consequently, the purpose of the present inven-tion is to provide a forced flow thin-layer chroma-tography device which responds better to the requirements of practice than the o~erpressure devices used up to the present, especially in that it has the advantage of requiring no prior preparation of the (rigid or flexible) chromatography plate and of not leading to the develop-ment of a vapour phase and in that it is particularly simple to use.
The subject of the present invention is a forced flow development thin-layer chromatography device, characterised in that it comprises a development chamber, in combination with means for reducing the pressure in the said chamber, which chamber contains at least one chromatography plate covered with a layer of suitable adsorbant and means for hermetically covering the said plate(s).
According to an advantageous embodiment of the said device, the said pressure-reducing means are coupled to a means for adjusting a descending pressure gradient.
According to a preferred arrangement of this embodiment, the said pressure-reducing means is especially a vacuum pump.
The means for adjusting the descending pressure gradient can be represented by a suitable obturator and, in a non-limiting way, by a micrometer screw coupled to a pulse motor.
. .
4 ~
According to another advantageous embodiment of the said device,-the means for hermetically covering the chromatography plate consist of a membrane which is hydrophobic and Lmpermeable to the migration solvents.
5According to an ad~antageous arrangement of this embodiment, the said membrane consists of a polyethylene foam containing closed cells.
The said polymer membrane is either of identical size to the chromatography plate or slightly smaller than the latter.
This device unexpectedly creates a homogeneous pressure reduction in the development chamber, and consequently in the adsorbent layer, and thus causes the polymer membrane to be firmly applied to the chromatographic support.
This device has a certain number of advantages with respect to OPTLC, as well as its simplicity of use:
- the vacuum created in the development chamber, especially using a single pump, makes it possible at the same time to:
homogeneously apply the polymer membrane to the chromatography plate, make possible migration of the mobile phase by "pulling", at constant velocity, and not by 'pushing' as is the case in the overpressure systems, which has the advantage of increasing the sensitivity of the chroma-tography dPvice and of reducing the migration time (obtaining results in a time which is markedly shorter than in the devices of the prior art) and 30be able to use any commercial chromatographic plate.
Together, these characteristics, which are specific to the device in accordance with the invention, additionally make it possible:
35- to avoid development of the vapour phase of the plate, and - to degas the pla~e.
.
Such a device, by avoiding development of a vapour phase, makes possible a development corresponding to a linear phenomenon and offers markedly improved separation results, as is shown in applying the formula Z = kt, in which Z: migration distance, k: constant, t:
migration time.
The advantages then obtained are:
- lengthening of the separation distance and - the possibility of optimising pulling by adjusting the descending pressure gradient, at constant temperature, but homogeneous for the layer of adsorbant, of the ~P = f(t) type.
The linear variation form ~P = a(t) + b cor-responds to an improved progression of the mobile phase and thu~ to a better separation. The migration velocity is dependent on the applied pressure variation (gradient) and is then influenced only by the density and the viscosity of the mobile phase and the temperature of the development chamber.
According to another advantageous embodiment of the said device, it can comprise a number of chromatographic plates separated by a suitable membrane, which makes posqible simultaneous analysis of a number of parameters and/or the separation of a large number of ; 25 solutes in a single development.
The subject of the present invention is also a process for detection and/or for quantitative determina-tion by thin-layer chromatography, in a chromatographic development chamber containing a suitable chromatography plate covered with a layer of suitable adsorbant, charac-terised in that the migration of the mobile phase through the stationary phase is carried out by pulling, by applying, in the said development chamber, a homogeneous reduced pressure in said adsorbent layer.
According to an advantageous method of implemen-tation of the said process, the said reduced pressure is applied in the form of a pressure gradient.
' ; ' "--' ' . ~
As well as the above arrangements, the invention also includes other arrangements which will emerge from the description herein~elow.
The invention will be better understood using the additional description hereinbelow, which refers to examples for implementation of the process in accordance with the invention and to a detailed description of the device of the invention, with reference to the appended drawings in which~
- Figure 1 represents a perspective view of a preferred embodiment of the device in accordance with the invention containing a plate;
- Figure 2 represents a cross-sectlon of the embodiment of Figure l;
t 15 - Pigure 3 represents a view illustrating the operation of a device in accordance with the invention;
- Figure 4 represents a curve which shows the influence of pressure on the distance covered by the solvent;
- Figure 5 represents another view illustrating the operation of a device in accordance with the inven-tion comprising a means for adjusting the descending pressure gradient.
It must be understood, however, that these drawings and the corresponding descriptive parts are given solely by way of illustration of the subject of the in~ention, of which they constitute in no way a lLmi-tation.
Reference is first made to Figures 1 to 4, which illustrate an em~odiment of the device in accordance with the invention.
Figure 1 represents a perspective view of a pre-ferred embodiment of the device in accordance with the invention, comprising a chromatographic development chamber 10 delimited, in the embodiment represented, by th~ walls 11 and 12; the lower wall (wall 12) is advan-tageously combined with a caliper 13, which makes it possible to obtain a development chamber which is larger . ~ : . . . .. .
, .. . . . , . .. . - . : . . :
- or smaller in size depending upon requirements. The development chamber 10 is combined with pressure-reducing means, not represented, at the orifice 14.
The development chamber 10 contains, in the embodiment represented, a chromatographic plate 20 which is covered with an adsorbent layer suitable for the thin-layer chromatography to be carried out and a polymeric membrane 30 which is applied hermetically and homogene-ously to the said adsorbent layer during application of reduced pressure.
The walls 11 and 12 are fastened by any suitable mean~.
The chromatographic plates 20 are preferably, and in a non-limiting way, glass plates precoated with a silica gel ("~ieselgel" G60 F254, ~erck).
The polymeric membrane 30 is prefera~ly, but in a non-limiting way, a hydrophobic polyethylene membrane containing closed cells ( Recticel , France; dimensions 180 x 200 x 3 mm) which provides a slight pressure on the surface of the chromatographic plate 20; thus, the only passage route for the solvent during chromatography is inside the adsorbent layer.
The physicochemical characteristics of this polyethylene membrane are the following:
Table I
Water absorption capacity 0%
Ignitability inflammable Density 30 kg/m Loss on drying 1%
Stability (72 h) at high temperature (llO~C) no change at low temperature (-18C) no change to W light (350 nm, 32C) no change Table II below specifies the behaviour of this membrane with respect to solvents which are commonly used in TLC.
``' - 8 -- Table II
SOLVENTCOEFFICIENT OF EXPANSION -:
5 water < 5%
methanol 14% :~ :
ethanol 16~
n-butanol 16%
10 acetone 16%
hep_ane 15~
toluene 32%
chloroform 29 ethyl acetate 16%
15 diethyl ether 13%
hexane 16%
acetonitrile 16%
methyl ethyl ketone 16%
20 hydrochloric acid 20%
acetic acid 16%
formic acid < 5%
aqueous ammonia 17%
25 pyridine 16%
. .
Figure 2 represents a cross-section of the embodiment of Figure 1, in the closed position.
: Figure 3 represents a perspective view of the embodiment of Figure 1.
3a The operation of such devices, illustrated in Figure 3, is as follows:
After deposition of the samples to be analysed on the adsorbent layer of the chromatography plate 20, the latter is introduced into the development ch~her 10, so that the part which contains the sample deposits is on the side opposite (side A of the device in accordance with the invention) orifice 14 (side B of the device in accordance with the invention), the side A of the device in accordance with the invention, hermetically closed using closing means 15, is immersed in a reservoir 50 containing the mobile phase 51 (suitable solvent) and the development chamber is connected to a liquid jet vacuum pump via the orifice 14.
This device creates a homogeneous pressure reduction in the development chamber and more particu-larly in the adsorbent layer and make~ it possible to carry out a TLC at reduced pressure, also called Vacuum P 1 ~nar Chrom~tography ( VPC ) .
Reference is also made to Figure 5, which illus-trates an embodiment of the device according to the invention, comprising a means for adjusting the descend-ing pressure gradient; the device of Figure 5 comprises a development chamber 10, such as described in Figure 3, in side view, combined, in the embodiment represented, with pressure-reducing means at the orifice 14. These : pressure-reducing means comprise a vacuum source represented by the vacuum pump 42, connected to the device 10 via the pipe 44; the said vacuum source is also connected to a means for adjusting the descending pressure gradient represented, in this embodiment, by a micrometer screw 4~ coupled~to a pulse motor 41, which makes it possible to adjust the obturation of an air inlet 43 placed on the course of the pipe 44.
The motor 41 is advantageously programmable, both for the obturation speed of the vacuum circuit (pipe 44 and air inlet 43) and for the method of selection of obturation speed variation (linear or exponential) ~xample 1: Influence of reduced pre~ure on the chroma-tography.
The distances covered by a solvent (methanol) at different points of the migration are measured (Z). These distances are represented in Figure 4 as a function of time. The curve ~ (5 mm of Hg) comprises two parts:
producing a pressure of 5 mm of Hg in 3 minutes and then maintaining reduced pressure constant at 5 mm of Hg by ~topping the reduced pressure gradient. It can be observed that the linear reduced pressure gradient . . .
corresponds to a linear variation in the distance covered ~ -as a function of time. At constant pressures, the dis-tance no longer advances in a controlled way. These observations can also be carried out regarding the curve ~ (1 mm of Hg~. It can be seen that the velocity of the mobile phase in the development chamber at reduced pressure is a function of the pressure (P) and that this relation is not linear. Only a progressive pressure reduction ~P makes it possible to have a linear relation, 10 if ~P=f (t). The reduced pressure device thus makes possible the progressive reduction of P during the analysis to ensure the linearity of the equation Z = kt.
Example 2: Quantitative determination of sodium pyruvate using the device in accordance with the invention.
5 Depositions of 5 ~1 of a 1 g/l sodium pyruvate solution are applied to a 200x200 mm, non-fluorescent silica TLC plate (Merck, Darmstadt, FRG).
The plate is placed in a device in accordance with the invention, as described above.
The mobile phase consists of:
Isopropanol 70 Demineralised water 35 The migration is carried out under gentle reduced pressure, corresponding to a final reduced pressure of 1 mm of ~g, obtained by a gradient of 0 to 1 mm of ~g for 30 minutes.
Location is achieved using the process described \ in European Patent Application No. 90910775~.4, by derivation under pressure with a bromocresol purple solution:
Bromocresol purple 40 mg 50% ethanol 100 ml Concentrated NaO~ q.s. p~ 10 At the same time, a TLC migration is carried out under the same operating conditions.
In both cases, a demixing region appears but with a markedly smaller width when the chromatography is carried out using the device in accordance with the - - , ~ :- - , .
.. . . . . .
invention; moreover, the sodium pyruvate spots are situated above the demixing region for TLC, in contrast to the chromatography carried out with the device accord-ing to the invention.
The Rf values of the spots are 0.85 in both cases.
~xample 3: Parameter: migratio~ rate.
A quantitative determination of sodium pyruvate is carried out using the device i~ accordance with the invention (cf. Example 2); the migration time is 30 minutes (+ 1 minute).
For developments carried out under the same operating conditions, but with a conventional TLC device, the migration lasts 50 minutes (+ 2 minutes).
As emerges from the above, the invention is in no way limited to those of its methods of implementation, embodiment and application which have been more explicitly described here; on the contrary, it embraces all the variants of them which can occur to the pràctitioner of the subject, without departing from the context or from the scope from the present invention.
-'',- ~ :
~ device for implementing this method is desc-ribed especially as follows in Patent Application 2,483,248 or Patent Application 2,580,406: a TLC plate placed on a glass or plastic support, comprising a lower part and an upper part, is completely covered on its upper part with a plastic membrane (pressure membrane~, which tightly fits its surface due to the gas pressure applied in the space delimited by the membrane and the upper part of the support unit of the device.
The gas inlet a~d the pressure gauge (manometer, for example), in combination with a syringe for intro-ducing the solvent and an inlet for introducing the sample under pressure, are in the upper part of the support unit. In this device, the external pressure on the mem~rane must always be greater than the pressure exerted on the eluent (solventJ and thus leads to an overpressure. The upper part and the lower part of the support unit are held together by means of clips.
~his device and the process which follows from it have a certain number of advantages and especially make it possi~le to optimise the separation conditions by adjusting the eluent flow rate. ~owever, OPTLC never-theless has a certain number of disadvantages:
- the necessity to prepare the chromatography plates, prior to their use, to create 4 sealed joins;
- the presence of a vapour phase in the adsorbent layer during development; indeed, such a vapour phase has the disadvantage o~ altering the selectivity (separation power) of the mobile phase, especially in the case of mixtures of solvents, of reducing the distance covered by the solutes (R~) from 20 to 40%, of reducing the maximum R~, beyond which no separation is possible, to values of between 0.6 and 0.8 (formation of a virtual front) and also of leading to demixing of the mobile phase and modification of the composition of the latter;
- the necessity especially for two pressure pumps, an eluent pump and a pump for pressurising the plate via the pressure membrane, which adheres hermeti-cally to the said plate.
- Consequently, the purpose of the present inven-tion is to provide a forced flow thin-layer chroma-tography device which responds better to the requirements of practice than the o~erpressure devices used up to the present, especially in that it has the advantage of requiring no prior preparation of the (rigid or flexible) chromatography plate and of not leading to the develop-ment of a vapour phase and in that it is particularly simple to use.
The subject of the present invention is a forced flow development thin-layer chromatography device, characterised in that it comprises a development chamber, in combination with means for reducing the pressure in the said chamber, which chamber contains at least one chromatography plate covered with a layer of suitable adsorbant and means for hermetically covering the said plate(s).
According to an advantageous embodiment of the said device, the said pressure-reducing means are coupled to a means for adjusting a descending pressure gradient.
According to a preferred arrangement of this embodiment, the said pressure-reducing means is especially a vacuum pump.
The means for adjusting the descending pressure gradient can be represented by a suitable obturator and, in a non-limiting way, by a micrometer screw coupled to a pulse motor.
. .
4 ~
According to another advantageous embodiment of the said device,-the means for hermetically covering the chromatography plate consist of a membrane which is hydrophobic and Lmpermeable to the migration solvents.
5According to an ad~antageous arrangement of this embodiment, the said membrane consists of a polyethylene foam containing closed cells.
The said polymer membrane is either of identical size to the chromatography plate or slightly smaller than the latter.
This device unexpectedly creates a homogeneous pressure reduction in the development chamber, and consequently in the adsorbent layer, and thus causes the polymer membrane to be firmly applied to the chromatographic support.
This device has a certain number of advantages with respect to OPTLC, as well as its simplicity of use:
- the vacuum created in the development chamber, especially using a single pump, makes it possible at the same time to:
homogeneously apply the polymer membrane to the chromatography plate, make possible migration of the mobile phase by "pulling", at constant velocity, and not by 'pushing' as is the case in the overpressure systems, which has the advantage of increasing the sensitivity of the chroma-tography dPvice and of reducing the migration time (obtaining results in a time which is markedly shorter than in the devices of the prior art) and 30be able to use any commercial chromatographic plate.
Together, these characteristics, which are specific to the device in accordance with the invention, additionally make it possible:
35- to avoid development of the vapour phase of the plate, and - to degas the pla~e.
.
Such a device, by avoiding development of a vapour phase, makes possible a development corresponding to a linear phenomenon and offers markedly improved separation results, as is shown in applying the formula Z = kt, in which Z: migration distance, k: constant, t:
migration time.
The advantages then obtained are:
- lengthening of the separation distance and - the possibility of optimising pulling by adjusting the descending pressure gradient, at constant temperature, but homogeneous for the layer of adsorbant, of the ~P = f(t) type.
The linear variation form ~P = a(t) + b cor-responds to an improved progression of the mobile phase and thu~ to a better separation. The migration velocity is dependent on the applied pressure variation (gradient) and is then influenced only by the density and the viscosity of the mobile phase and the temperature of the development chamber.
According to another advantageous embodiment of the said device, it can comprise a number of chromatographic plates separated by a suitable membrane, which makes posqible simultaneous analysis of a number of parameters and/or the separation of a large number of ; 25 solutes in a single development.
The subject of the present invention is also a process for detection and/or for quantitative determina-tion by thin-layer chromatography, in a chromatographic development chamber containing a suitable chromatography plate covered with a layer of suitable adsorbant, charac-terised in that the migration of the mobile phase through the stationary phase is carried out by pulling, by applying, in the said development chamber, a homogeneous reduced pressure in said adsorbent layer.
According to an advantageous method of implemen-tation of the said process, the said reduced pressure is applied in the form of a pressure gradient.
' ; ' "--' ' . ~
As well as the above arrangements, the invention also includes other arrangements which will emerge from the description herein~elow.
The invention will be better understood using the additional description hereinbelow, which refers to examples for implementation of the process in accordance with the invention and to a detailed description of the device of the invention, with reference to the appended drawings in which~
- Figure 1 represents a perspective view of a preferred embodiment of the device in accordance with the invention containing a plate;
- Figure 2 represents a cross-sectlon of the embodiment of Figure l;
t 15 - Pigure 3 represents a view illustrating the operation of a device in accordance with the invention;
- Figure 4 represents a curve which shows the influence of pressure on the distance covered by the solvent;
- Figure 5 represents another view illustrating the operation of a device in accordance with the inven-tion comprising a means for adjusting the descending pressure gradient.
It must be understood, however, that these drawings and the corresponding descriptive parts are given solely by way of illustration of the subject of the in~ention, of which they constitute in no way a lLmi-tation.
Reference is first made to Figures 1 to 4, which illustrate an em~odiment of the device in accordance with the invention.
Figure 1 represents a perspective view of a pre-ferred embodiment of the device in accordance with the invention, comprising a chromatographic development chamber 10 delimited, in the embodiment represented, by th~ walls 11 and 12; the lower wall (wall 12) is advan-tageously combined with a caliper 13, which makes it possible to obtain a development chamber which is larger . ~ : . . . .. .
, .. . . . , . .. . - . : . . :
- or smaller in size depending upon requirements. The development chamber 10 is combined with pressure-reducing means, not represented, at the orifice 14.
The development chamber 10 contains, in the embodiment represented, a chromatographic plate 20 which is covered with an adsorbent layer suitable for the thin-layer chromatography to be carried out and a polymeric membrane 30 which is applied hermetically and homogene-ously to the said adsorbent layer during application of reduced pressure.
The walls 11 and 12 are fastened by any suitable mean~.
The chromatographic plates 20 are preferably, and in a non-limiting way, glass plates precoated with a silica gel ("~ieselgel" G60 F254, ~erck).
The polymeric membrane 30 is prefera~ly, but in a non-limiting way, a hydrophobic polyethylene membrane containing closed cells ( Recticel , France; dimensions 180 x 200 x 3 mm) which provides a slight pressure on the surface of the chromatographic plate 20; thus, the only passage route for the solvent during chromatography is inside the adsorbent layer.
The physicochemical characteristics of this polyethylene membrane are the following:
Table I
Water absorption capacity 0%
Ignitability inflammable Density 30 kg/m Loss on drying 1%
Stability (72 h) at high temperature (llO~C) no change at low temperature (-18C) no change to W light (350 nm, 32C) no change Table II below specifies the behaviour of this membrane with respect to solvents which are commonly used in TLC.
``' - 8 -- Table II
SOLVENTCOEFFICIENT OF EXPANSION -:
5 water < 5%
methanol 14% :~ :
ethanol 16~
n-butanol 16%
10 acetone 16%
hep_ane 15~
toluene 32%
chloroform 29 ethyl acetate 16%
15 diethyl ether 13%
hexane 16%
acetonitrile 16%
methyl ethyl ketone 16%
20 hydrochloric acid 20%
acetic acid 16%
formic acid < 5%
aqueous ammonia 17%
25 pyridine 16%
. .
Figure 2 represents a cross-section of the embodiment of Figure 1, in the closed position.
: Figure 3 represents a perspective view of the embodiment of Figure 1.
3a The operation of such devices, illustrated in Figure 3, is as follows:
After deposition of the samples to be analysed on the adsorbent layer of the chromatography plate 20, the latter is introduced into the development ch~her 10, so that the part which contains the sample deposits is on the side opposite (side A of the device in accordance with the invention) orifice 14 (side B of the device in accordance with the invention), the side A of the device in accordance with the invention, hermetically closed using closing means 15, is immersed in a reservoir 50 containing the mobile phase 51 (suitable solvent) and the development chamber is connected to a liquid jet vacuum pump via the orifice 14.
This device creates a homogeneous pressure reduction in the development chamber and more particu-larly in the adsorbent layer and make~ it possible to carry out a TLC at reduced pressure, also called Vacuum P 1 ~nar Chrom~tography ( VPC ) .
Reference is also made to Figure 5, which illus-trates an embodiment of the device according to the invention, comprising a means for adjusting the descend-ing pressure gradient; the device of Figure 5 comprises a development chamber 10, such as described in Figure 3, in side view, combined, in the embodiment represented, with pressure-reducing means at the orifice 14. These : pressure-reducing means comprise a vacuum source represented by the vacuum pump 42, connected to the device 10 via the pipe 44; the said vacuum source is also connected to a means for adjusting the descending pressure gradient represented, in this embodiment, by a micrometer screw 4~ coupled~to a pulse motor 41, which makes it possible to adjust the obturation of an air inlet 43 placed on the course of the pipe 44.
The motor 41 is advantageously programmable, both for the obturation speed of the vacuum circuit (pipe 44 and air inlet 43) and for the method of selection of obturation speed variation (linear or exponential) ~xample 1: Influence of reduced pre~ure on the chroma-tography.
The distances covered by a solvent (methanol) at different points of the migration are measured (Z). These distances are represented in Figure 4 as a function of time. The curve ~ (5 mm of Hg) comprises two parts:
producing a pressure of 5 mm of Hg in 3 minutes and then maintaining reduced pressure constant at 5 mm of Hg by ~topping the reduced pressure gradient. It can be observed that the linear reduced pressure gradient . . .
corresponds to a linear variation in the distance covered ~ -as a function of time. At constant pressures, the dis-tance no longer advances in a controlled way. These observations can also be carried out regarding the curve ~ (1 mm of Hg~. It can be seen that the velocity of the mobile phase in the development chamber at reduced pressure is a function of the pressure (P) and that this relation is not linear. Only a progressive pressure reduction ~P makes it possible to have a linear relation, 10 if ~P=f (t). The reduced pressure device thus makes possible the progressive reduction of P during the analysis to ensure the linearity of the equation Z = kt.
Example 2: Quantitative determination of sodium pyruvate using the device in accordance with the invention.
5 Depositions of 5 ~1 of a 1 g/l sodium pyruvate solution are applied to a 200x200 mm, non-fluorescent silica TLC plate (Merck, Darmstadt, FRG).
The plate is placed in a device in accordance with the invention, as described above.
The mobile phase consists of:
Isopropanol 70 Demineralised water 35 The migration is carried out under gentle reduced pressure, corresponding to a final reduced pressure of 1 mm of ~g, obtained by a gradient of 0 to 1 mm of ~g for 30 minutes.
Location is achieved using the process described \ in European Patent Application No. 90910775~.4, by derivation under pressure with a bromocresol purple solution:
Bromocresol purple 40 mg 50% ethanol 100 ml Concentrated NaO~ q.s. p~ 10 At the same time, a TLC migration is carried out under the same operating conditions.
In both cases, a demixing region appears but with a markedly smaller width when the chromatography is carried out using the device in accordance with the - - , ~ :- - , .
.. . . . . .
invention; moreover, the sodium pyruvate spots are situated above the demixing region for TLC, in contrast to the chromatography carried out with the device accord-ing to the invention.
The Rf values of the spots are 0.85 in both cases.
~xample 3: Parameter: migratio~ rate.
A quantitative determination of sodium pyruvate is carried out using the device i~ accordance with the invention (cf. Example 2); the migration time is 30 minutes (+ 1 minute).
For developments carried out under the same operating conditions, but with a conventional TLC device, the migration lasts 50 minutes (+ 2 minutes).
As emerges from the above, the invention is in no way limited to those of its methods of implementation, embodiment and application which have been more explicitly described here; on the contrary, it embraces all the variants of them which can occur to the pràctitioner of the subject, without departing from the context or from the scope from the present invention.
-'',- ~ :
Claims (8)
1. Forced flow development thin-layer chromatography device, characterised in that it comprises a development chamber (10), in combination with means for reducing the pressure in the said chamber, which chamber contains at least one chromatography plate (20) covered with a layer of suitable adsorbent (21) and means (30) for hermeti-cally covering the said plate(s).
2. Device according to Claim 1, characterised in that the said pressure-reducing means (42, 44) are coupled to a means for adjusting a descending pressure gradient (40, 41, 43).
3. Device according to Claim 1 or Claim 2, charac-terised in that the said pressure-reducing means is a vacuum pump (42).
4. Device according to any one of Claims 1 to 3, characterised in that the means (30) for hermetically covering the chromatography plate consist of a membrane which is hydrophobic and impermeable to the migration solvents.
5. Device according to Claim 4, characterised in that the said membrane consists of a polyethylene foam containing closed cells.
6. Device according to any one of Claims 1 to 5, characterised in that it comprises a number of chromato-graphic plates separated respectively by a suitable membrane.
7. Process for detection and/or for quantitative determination by thin-layer chromatography, in a chrom-atographic development chamber containing a suitable chromatography plate covered with a layer of suitable adsorbent, characterised in that the migration of the mobile phase through the stationary phase is carried out by pulling, by applying, in the said development chamber, a homogeneous reduced pressure in the said adsorbent layer.
8. Process for detection and/or quantitative deter-mination by thin-layer chromatography according to Claim 7, characterised in that the said reduced pressure is applied in the form of a pressure gradient.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR9103849A FR2674631B1 (en) | 1991-03-29 | 1991-03-29 | METHOD AND DEVICE FOR PLANAR CHROMATOGRAPHY USING REDUCED PRESSURE AS A FORCE FLOW PRINCIPLE. |
| FR91/03849 | 1991-03-29 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2083939A1 true CA2083939A1 (en) | 1992-09-30 |
Family
ID=9411276
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002083939A Abandoned CA2083939A1 (en) | 1991-03-29 | 1992-03-27 | Thin-layer chromatography process and device bringing into play a reduced pressure as the forced flow means |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0532733B1 (en) |
| JP (1) | JPH05508024A (en) |
| AT (1) | ATE161331T1 (en) |
| CA (1) | CA2083939A1 (en) |
| DE (1) | DE69223567T2 (en) |
| FR (1) | FR2674631B1 (en) |
| HU (1) | HUT63702A (en) |
| RU (1) | RU2124200C1 (en) |
| WO (1) | WO1992017777A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109298126B (en) * | 2018-11-15 | 2020-08-25 | 西安医学院 | Panel paving device and panel paving method of thin-layer chromatographic plate |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE842863C (en) * | 1951-01-12 | 1952-07-03 | Siegmund & Co Dr | Chromatographic and electrophoretic apparatus and methods, respectively |
| US3629098A (en) * | 1968-09-27 | 1971-12-21 | Toyo Roshi Co Ltd | Continuous flow, preparative thin-layer chromatograph |
| GB1262493A (en) * | 1969-06-11 | 1972-02-02 | Pye Unicam Ltd | Improvements in or relating to thin layer chromatography |
| US3864250A (en) * | 1973-03-16 | 1975-02-04 | Regis Chem Co | Liquid-solid adsorption chromatography |
| EP0060709B1 (en) * | 1981-03-17 | 1987-09-09 | Varex Corporation | Method and system for multi-dimensional real-time chromatography |
| DE3824896A1 (en) * | 1988-07-22 | 1990-01-25 | Wolfgang Heisig | Method and apparatus for sample analysis in thin layer chromatography |
-
1991
- 1991-03-29 FR FR9103849A patent/FR2674631B1/en not_active Expired - Fee Related
-
1992
- 1992-03-27 DE DE69223567T patent/DE69223567T2/en not_active Expired - Fee Related
- 1992-03-27 RU RU92016439A patent/RU2124200C1/en active
- 1992-03-27 JP JP92508591A patent/JPH05508024A/en active Pending
- 1992-03-27 EP EP92909051A patent/EP0532733B1/en not_active Expired - Lifetime
- 1992-03-27 WO PCT/FR1992/000275 patent/WO1992017777A1/en active IP Right Grant
- 1992-03-27 AT AT92909051T patent/ATE161331T1/en not_active IP Right Cessation
- 1992-03-27 HU HU9203749A patent/HUT63702A/en unknown
- 1992-03-27 CA CA002083939A patent/CA2083939A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| ATE161331T1 (en) | 1998-01-15 |
| EP0532733B1 (en) | 1997-12-17 |
| RU2124200C1 (en) | 1998-12-27 |
| FR2674631B1 (en) | 1994-07-22 |
| DE69223567D1 (en) | 1998-01-29 |
| DE69223567T2 (en) | 1998-07-09 |
| EP0532733A1 (en) | 1993-03-24 |
| HU9203749D0 (en) | 1993-04-28 |
| JPH05508024A (en) | 1993-11-11 |
| FR2674631A1 (en) | 1992-10-02 |
| WO1992017777A1 (en) | 1992-10-15 |
| HUT63702A (en) | 1993-09-28 |
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| Date | Code | Title | Description |
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