CHROMATOGRAPHIC SHEET OF LAYER FOR PRESSURIZED LAYER CHROMATOGRAPHIC APPARATUS

Abstract: Chromatographic sheet of layer for pressurized layer chromatographic apparatus, for the development of linear chromatogram, which is characterized by sealing the sorbent layer at the edges of the sheet of layer applied conventionally with normal or fine-grained sorbent layer, and the sorbent layer is removed in one or several zones near the place of solvent admission, or one or several baffle plates or wires are arranged behind the sample application place or places, and -if necessary- it is provided with conventionally formed concentrating zone on the part from the direction of the solvent admission. The developing solvent composite does not escape at the edges of the sheet of layer in case of overpressure, and it advances with straight front.

Description: CHROMATOGRAPHIC SHEET OP LAYER FOR PRESSURIZED LAYER CHROMATOGRAPHIC APPARATUS The invention relates to specially prepared chro¬ matographic sheet of layer favourably used in the pressuriz¬ ed layer-chro atographic apparatuses, for the development of linear chromatogram. The layer chromatography as liquid chromatography of planar arrangement is a method whereby a mobile phase (solvent composite) travels on the stationary phase (on sol¬ vent composite or sorbent) and carries one or several sub¬ stances at varying speed. The most significant process of those, taking place meanwhile is the distribution of the travelling compounds between the two phases. Drawback of the classical layer-chromatography is the long developing time, and due to the short sheet of layer, the separation is not prefect, the theoretical number of plates is limited. Significant progress was made by development of the high performance thin layer chromatography (HPTLC) (A. Zkatkis and R.Ξ.' Kaiser: High Performance Thin-layer Chromatography, Elsevier, 1977), which contrary to the clas¬ sical layer chromatography results in better dissolution within shorter developing time and at shorter distance. However, the most up-to-date technicςue is the so-called pressurized (sealed) layer chromatography (Hunga¬ rian Patent Ho. 173 749) realizable equally on thin and thick layers, and the separation is the fastest. Substance of the pressurized layer chromatography is that the solvent system is flown on the sorbent layer with overpressure in a closed system. The sorbent layer is complet.ely covered suitably with elastic foil, and which is pressed cushion- -like to the sorbent layer v/ith the use of external pressure. -% The cushion pressure (2-10 bar) shall always be higher than the flow pressure (1-8 bar) of the liquid composite In this so-called pressurized ultra icron chamber a bett separation can be attained within a very short time (1-1 5 minutes), than in the earlier known chromatographic meth of planar arrangement. An apparatus was constructed for the development of both the concentric and linear chroma tograms. However, it is a fundamental problem at the li 10 alternative of the pressurized layer chromatography, tha as a result of the overpressure, the solvent composite e capes at the edges of the chromatographic layer, and tha the solvent front is not straight, since concentric, sem -circular, and quarter-circular front lines arise around l J *ϊ the solvent admission, e.g. in case of solvent admission one point. At working out the invention, the development of such chromatographic sheet of layer was aimed at, hich is applicable in the pressurized layer-chromatograρhi*c 20 apparatus without escape of the developing solvent compo at the edges, on which the solvent composite travels wit straight front. The invention is based on the recognition, tha if the sorbent layer is suitably sealed at the edges of 25 the sheet of layer, then the solvent can not escape in c of overpressure. Furthermore the invention is based on the reco tion, that if the sorbent layer at and/or near the place of admitting the solvent composite is removed from the 0 carrier plate in one or several thin zones, or thin baff plate, or plates, or wires are placed directly in front the place of admission, i.e. mini-solvent composite cham are formed, then the solvent composite will run with straight front-line. QMPI Sealing of the sorbent layer according to the invention is realized v/ith impregnation, or the sorbent layer is shrunk with the use of powdered glass, whereby varying shrinkage conditions are ensured at the edges of the layer, and in the chromatographic field, or one or several parallel grooves are formed in the carrier plate, and the sorbent layers are placed into these grooves, whereby the carrier plate itself seals the edges, or on case of using ceramic plate, its edges are glazed. The impregnating or glazing materials shall be insoluble in the solvent used for the chromatography, they shall not be altered as a result of the overpressure of the solvent composite, and under the given temperature condition, and no chemical reaction should occur v/ith the sorbent layer or foil, sealing the sorbent layer from above. Preferably paraffin or synthetic solutions, or suspensions, water-glass (sodium silicate) may be used as impregnating material. The edges of the ceramic plates sy be coated with white tinlead glaze. In case of shrinkage with pov/dered glass, varying quality of powdered glass is used on the edges, but the use of other additives on the edges is also suitable. For one-dimensional development it is sufficient to seal the two edges of the layer, and that of the side opposite the development direction of the front. For two- -di ensional development, however, it is necessary to seal the layer on all the four sides. For the two-directional, linear development with intermediate solvent-admission - it is sufficient to seal the two opposite edges - suitably the longer ones of the sheet of layer. Width of the sealing layer may vary ' between 0.05 and 3 cm. Most favourable is the 1 cm or thinner layer. Length of the layer is the same as that of the chromato¬ graphic plate. ____ . QMPI / , WIPO ,- The straight front line of the developing sol¬ vent composite according to the invention is ensured in such a manner, that the sorbent layer is removed in a width of 0.1 - 0.5 mm in one or several zones at or near the place of admitting the solvent composite, as to have the zone of the carrier plate sorbent-free. Maximum lengt of the so-formed grooves is the same as those of the sor¬ bent layer retained after coating of the edges, but prefe ly shorter by 1-20 mm on both sides (Fig. 1). The grooves may be formed by displacement in relation to each other (Fig. 2) . Maximum depth of the grooves shall not exceed the thickness of the sorbent layer. Their number may vary preferably between 1 and 4. In. the process according to t invention 0.05 - 1-0 m diameter wire or wires, or baffle plates can be arranged behind the place or places of samp application. Maximum length of the baffle plates or wires after coating of the edges is the same as that of the re¬ tained sorbent layer, but preferably shorter by 1-20 mm on both sides. The baffle plates or wires can be displace in relation to each other. Their number is preferably 1-4 Preferably gapless synthetic plate is used fixed by glazi to the sheet of layer. A so-called "concentration zone" can also be fo on the solvent admission part of the sheet of layer forme as described above, which means that a zone of another la is applied on the sorbent layer (Fig. 3) - The material of this layer may be for instance siliceous earth powdered glass, or synthetic powder, or cellite, i.e. rather carri than εorbents used for separation (e.g. aluminium oxide, silica gel) , but occasionally highly porous silica gel of wide pore range is useable against the low porosity, fine -graineα layer. The so-formed double layer facilitates th concentration of the sample. Preferably fine-grained (0.5 - 10 u ) , or nor¬ mal (10-40 /urn) sheet of layer can be used for the chroma¬ tographic sheet of layer according to the invention. Any sheet of layer available in the trade can be developed for the pressurized (sealed) layer-chromatography according to the invention. However, the sorbent layer may be prepared domestica!!,*-, too. The developing solvent composite does not escape on the edges of the sheet of layer according to the inven- tion in case of overpressure and it advances with straicght front. The invention is illustrated with the following examples: Example 1 110 ml 0.85 %. atery solution of relatively highly polymerized homopolymeric polyacrylamide is added to 32 g silica gel powder of 4-8 /u grain size and βO A inside diameter. After thorough mixing the 0.25 inm thick suspen¬ sion is applied on degreased, 20 x 20 cm, 1,5 mm thick glass panels according to the known method. The layers dried at room temperature are of good, mechanical strength even after activation. Edges of the. so-obtained and activated aheet of layers are impregnated in a width of 10 mm, by dipping on three sides into commercial paraffin melted at 130 C. For directing the solvent, 0.2 mm diameter steel wire is placed in front of the solvent admission, as shown in Fig. 1. V/ith use of methylene chloride solvent, authentic dye materials (butter-yellow, Sudan G and indophenol) can be v/ell separated in the so-called pressurized ultramicron chanber at 1.0LPa cushion pressure, with 10 minute develop¬ ing time, at a distance of 15 cm. Example 2 The procedure is the same as in Example 1, but in place of 32 g silica gel powder, 30 g neutral alumi¬ nium oxide of 15-20 /urn grain size is used, applied in a thickness of 0.3 mm. Example 3 100 ml 1.8 j. watery solution of the polymersi of acrylamide and acrylic acid anionogen mixture of low polymerization degree is added to silica gel powder of 4-5 um grain size and 60 A inside diameter. After thoro mixing the suspension is applied in a thickness of 0.3 m onto 20 x 40 cm. 0.4 mm thick polyterephthalate foil. Th layers dried at 75 C have adequate strength even after activation. Immediately after the application arid before the previously described drying, for the subsequent solv control 2 mm wide, 0.3 mm thick and 185 mm long polytere thalate foil is placed in front of the intended place of solvent admission to 15 mm fron the odd impregnable edge of the plate, and at equal distance from the other tv/o ed to be impregnated (for schematic diagram see Fig. 1). Th plate sticks into the layer during the drying process. Edges (on three sides) of the so-obtained dried sheets of layer provided with control plate (foil) are impregnated by dipping into the 1.5 % watery solution of highly polymerized, non-ionogen polyacrylamide homopoly- merism in a width of 5 mm and dried at room temperature. On the so-formed sheet of layer Digital glyco- sides v/ere effectively separated in linear pressurized ultramicron chamber, with the use of butanon-2 developer on a distance of 35 cm, at 1.0 MPa cushion pressure, in 18.5 minutes. QMPI Example 4 The procedure is the same as in Example 2, but two grooves each of 3 ram are formed in the layer for the solvent control (see schematic diagram in Fig. 2). Example 5 100 ml 2 % watery solution of poly-vinyl-acetate is added to 26 g silica gel powder of 8-10 / m grain size and 60 A inside diameter. Simultaneosuly 30 ml 2 % watery poly-vinyl-acetate suspension is added to 8 g siliceous earth of 10-20 /u grain sizei The two systems are homoge¬ nized is separate electric homogenizer for one minute, then the obtained two suspensions are poured into two troughs separated proportionally in space and length, and applied to 0.2 mm thick 20 x 20 cm aluminium foil in the usual v/ay. Thus 0.25 nun thick, coupled two-zone sorbent layer is ob¬ tained, where width of the inactive zone (according to the separation of the trough in space and length): 40 mm (Fig. 3). Before drying of the sorbent layer, immediately after the application, 0.3 iπm thick, 1 mm wide, 160 mm long poly-terephthalate plate is placed, in front of the intended place of solvent admission to 15 mm from the odd, impregnable edge of-the plate from the direction of the in¬ active sorbent zone and at equal distance from the other two, parallel edges to be impregnated for the purpose of the subsequent solvent control. Then the layer is dried, and possibly activated. Lpregnation of the layer's edge in this case was performed as described in Example 2. On the so-formed layer provided with the so-called concentration zone, the components (εesquiterpenoids) of the volatile oil samples obtained from the inflorescence of the Hatricaria chamonmilla L. by steam destination were effecti¬ vely separated in linear pressurized chamber with bensol as solvent, at 0.7 MPa cushion pressure, with 20 ml/h solvent volumetric velocity on a distance of 15 cm, in 12 minutesj Example 6 The procedure is the same as in Example 4, but 7 g cellite powder of 10-20 u grain size is used instead of the 8 g siliceous earth. Example 7 36 g magnesium-silicate powder of 6-10 u grai size and 110 g 6 Be sodium silicate watery solution is homogenized in electric stuf -c usher, applied with the usual method on a suitably cleane 1.5 mm thick glass pa- nel, then the plates are stored in a chamber of 75 % rela tive moisture content for 2.5 hours, then removed and dri at room temperature. Thus adequately stable layer is obta ed. Edges of the so-prepared plates are impregnated as described in Example 1, and it can be used similarly f the effective separation of dye materials. Example 8 The procedure is the same as described in Examp 3, except.that the two opposite long edges of the layer a impregnated still before drying of the sorbent layer by dipping into the 1.5 % watery solution of the highly poly merized non-ionogen"polyacrylamide homopolymerism, and at the same time 1 mm thick groove is made in the centre of layer at the intended place of solvent admission for the subsequent solvent control and two-directional developmen then the layer is dried and used for development. Example 9 The procedure is the same as described in Examp 7 » except that the layer is made suitable for tv/o-dimensi development by impregnating it on the 4th side and well a the one-dimensional development and the solvent control i solved as shown in Figure 4. QMPI WIPO Example 10 6 grooves of 0.3 mm depth are formed in a 20 x 20 cm, 2 mm thick aluminium plate, into which paste made of polyamide 11 powder of 8-10 /u grain size is applied (25 g polyamide powder is mixed in 100 ml 1.5 % watery poly-vinyl- -acetate solution, followed with homogenization for 1 mi¬ nute). 15 mm wide barriers on the edges of the layer and 20 mm barriers on the side opposite the solvent-flov/ are formed, while the barriers separating the grooves from each other are formed to smooth surface of 10 mm width. In order to ensure the straight solvent-front, 16 x 3 x 0.3 mm synthe¬ tic foil (polyethylene) is arranged " in front of the six solvent admission openings, as shown in Fig. 5. Application of the samples took place at 12 mm from the solvent admis- sion opening. Example 11 The procedure is the same as in Example 1, but the applied silica gel contains manganese-activated sinc- -εilicate, as fluorescent material. QMPI - -figures Figure 1 : Schematic diagram of finished silica gel layer v/ith impregnated edge, including the operations. a. = silica gel zone impregnated with solid para fic b_ = places of sample application £ = place of solvent admission _ = steel wire Figure 2 : Schematic diagram of silica gel layer with i preg- nated edge. The solvent control is * ensured v/ith grooves. a , = impregnated silica gel layer zone b_ = grooves £ = solvent admission t = places of sample application Figure 3 : Silica gel layer with impregnated edge, provided ■ with concentrating zone. Figure 4 : Schematic diagram of magnesium-silicate layer impregnated on four sides. The solvent control is performed with synthetic foil in both dimensions, a = impregnated magnesium-silicate layer zone b_ = solvent admission £ = synthetic foil d_ = place of sample application Figure 5 : Schematic diagram of sorbent layer placed into grooves made in the carrier plate, a. = place of sample application b_ = synthetic foil £ = place of solvent admission _ = sorbent layer e = barriers formed from the carrier late. QMPI /,, IPO «,

Claims: WHAT WE CLAIM IS 1. Chromatographic sheet of layer for pressurized layer-chromatographic apparatus, for the development of li- near chromato ram, c h a r a c t e r i z e d i n that the sorbent layer is sealed at the edges of the sheet of layer applied conventionally with normal or fine grained sorbent layer, and the sorbent layer is removed in one or several zones at, and/or near the place of solvent admission, or one or several baffle plates or λvires are arranged behind the sample application place, or places, and - when required . it is provided with conventionally formed concentrating zo¬ ne on the part from the direction of the solvent admission. 2. Embodiment of the sheet of layer as claimed in. claim 1, c h a r a c t e r i z e d i n that the sor¬ bent layer is sealed by impregnation, or in case of ceramic plate v/ith coat of glaze. 3. Embodiment of the sheet of layer as claimed in claim 1, c h a r a c t e r i z e d i n that the sor- bent layer is shrunk with powdered glass in such a manner, that the shrinkage varies at the edges of the layer and in the chromatographic ' field. 4. Embodiment of the sheet of layer as claimed in claim 1, c h a r a c t e r i z e d i n that one or several grooves are formed in the carrier plate and the sorbent layer is e placed in these grooves. 5. Embodiment of the sheet of layer as claimed in claim 1, ch a r a c t e r i z e d i n that the width of the sealing layer is 0.05 - 3-.0 cm, preferably 0.1 - 1 cm. 6. Embodiment of the sheet of layer as claimed in claim 1, c h a r a c t e r i z e d i n that the sorbent layer is sealed v/ith paraffin or synthetic solutions, or suspensions, or water-glass, or white tin-lead glaze. QMPI /., WIPO .v,. 7. Embodiment, of the sheet of layer as claimed in claim 1, c h a r a c t e r i z e d i n that the sorbent layer is sealed at two, three or four sides.. 8. Embodiment of the sheet of layer as claimed in claim 1, c h a r a c t e r i z e d i n that the sorbent layer is removed in a 0.l - 5.0 mm wide zone, or zones and the maximum length of the zones is the same as that o ' " the sorbent layer retained after coating of the edges, preferably shorter by 1-20 mm. on both sides, and the depth shall not exceed the maximum thickness of the sorbent layer. 9. Embodiment of the sheet of layer as claimed in claim 1, and 8, c h a r a c t e r i z e d i n that 1-4 zones are formed, which - if necessary - may. be dis- placed in relation to each other. 10. Embodiment of the sheet of layer as claimed in claim 1, c h a a c t e r i z e d i n that widt and height of the baffle plate or plates arranged behind the sample application place or places is 0.05 - 1.00 mm, maximum length is the same as that of the sorbent layer retained after coating of the edges, preferably shorter by 1-20 mm on both sides. 11. Embodiment of the sheet of layer as claimed in claims 1 and 10, c h a r a c t e r i z e d i n tha the diameter of the wire or v/ires arranged behind the sam application place or places is 0.05 - 1.00 mm, maximum length is the same as that of the sorbent layer retained after coating of the edges, preferably shorter by 1-20 mm on both sides. 12. Embodiment of the sheet of layer as claimed in claims 1, 10 and 11, c h a r a c t e r i z e d i n that 1-4 baffle plates or v/ires are arranged, which may b displaced in relation to each other. 13. Embodiment of the sheet of layer as claimed in claim 1, c h a r a c t e r i z e d i n that siliceous earth, powdered glass or cellite are used for the inactive concentrating zone.

Inventors: KALASZ H, KNOLL J, LAK I, MINCSOVICS E, NAGY J, TETENYI P, TYIHAK E, ZOLTAN S
Applicants: KALASZ H, KNOLL J, LAK I, MINCSOVICS E, MUESZERIPARI MUEVEK LAB, NAGY J, TETENYI P, TYIHAK E, ZOLTAN S, LABOR MUESZERIPARIMUEVEK
Application year: 1980
Main classification: G01N31
IPC codes: G01N30, G01N31