WO2003091723A2

WO2003091723A2 - Device for controlled drying and derivatization in planar chromatography and its application

Info

Publication number: WO2003091723A2
Application number: PCT/SI2003/000012
Authority: WO
Inventors: Mirko Prosek; Irena Vovk; Alenka Golc Wondra; Janko Zmitek
Original assignee: Kemijski Institut
Priority date: 2002-04-26
Filing date: 2003-04-17
Publication date: 2003-11-06
Also published as: WO2003091723A3; DE10392483T5; AU2003262558A1; SI21199A

Abstract

The inventive device allows to substantially improve the reproducibility of measurements in quantitative thin-layer chromatography (TLC and HPTLC). Accuracy, sensitivity and reproducibility are improved by eliminating the uncontrolled band broadening resulting from diffusion, nonuniform vertical in-depth distribution of spots in the sorbent layer during drying, and nonhomogeneous derivatization conditions. The main characteristics of the inventive device are as follows: the heated air flow is directed over the sorbent layer in the direction of chromatogram development. The temperature of the heated air is preferably not constant but regularly oscillating between a minimum and a maximum value. The range between the minimum and the maximum temperature values, as well as the length of the oscillation interval, may be regulated using a dedicated program. The device also supports computerized drying and automated operation through integration into a complete system for processing and evaluating chromatograms and results.

Description

Device for Controlled Drying and Derivatization in Planar Chromatography and Its Application

Technical Field

The present invention pertains to the technical field of analytical chemistry; more specifically, the invention relates to a device for controlled drying and derivatization in planar chromatography and its application, allowing the various steps of thin-layer chromatography to be carried out in a reproducible and controlled manner. Accuracy, sensitivity and reproducibility are improved by eliminating the uncontrolled band broadening resulting from diffusion, nonuniform vertical in-depth distribution of spots in the sorbent layer during drying, and nonhomogeneous derivatization conditions.

Technical Problem

In performing quantitative analysis with thin-layer chromatography (TLC), adequate drying of developed chromatographic plates is considered a major problem, since many parameters of the analysis, such as sensitivity, accuracy and reproducibility of the results, significantly depend on this step of the process. Therefore, a device enabling drying and other operations subsequent to the development of the chromatograms (such as derivatization) to be performed in a controlled and technically appropriate way is in demand. State of the Art

In the world, not much attention is given to the step of drying chromatographic plates, said step being wrongly assumed not to constitute a major source of error.

Manufacturers develop devices for the application and development of chromatograms, leaving it up to the laboratories to decide on the drying and derivatization procedures .

For this reason, in laboratory practice common laboratory dryers and dryers similar to hair dryers (blow-dryers) are used, which do not ensure adequate drying. Hence, in the case when a blow-dryer is used, the heating of the plate is not uniform, which, in turn, generally leads to a nonuniform distribution of spots in the layer, possibly even to overheating, and consequently to the decomposition of single analyte components. In a laboratory dryer, on the other hand, there is a temperature gradient, meaning that the temperature is not uniform over the entire TLC plate, which is why nonuniform drying occurs and, moreover, a laminar air flow can not be ensured and directed over the plate in the same direction as the plates were developed, resulting in an uncontrolled band broadening in all directions .

Because drying with a blow-dryer or in a laboratory dryer entails low reproducibility and is frequently the cause of inaccurate or non-reproducible analysis results, various manufacturers have tried to address the problem of drying by contriving special heating plates with controlled temperature. Visually, these plates resemble electrical hot plates and are arranged in such a way that the temperature on the surface of the heating plate is as uniform as possible. However, a heating plate only conveys energy and does not remove the vapors of the evaporating mobile phase. Thus solvent vapors accumulate on the surface of the sorbent, leading, in turn, to horizontal band broadening and consequently to errors and/or non-reproducible results. Sometimes, the errors may also be ascribed to the random removal of vapors caused by the uncontrolled air flow passing over the working surface of the chromatographic plate. Therefore, even employment of heating plates does not warrant sufficiently reproducible and accurate results .

In derivatization, the main drawback of heating plates is nonuniform temperature over the surface of the plate (sorbent), primarily because of the uncontrolled transfer of heat to the environment. This gives rise to differences in the derivatization reactions between various regions of the plate, thus reducing accuracy and reproducibility.

In laboratory practice, the problem of considerably dispersed results is addressed by applying a larger array of parallels of the same sample and standard onto the plate and by using a larger number of chromatographic plates, in other words, by averaging a larger number of results. All these factors significantly reduce the utility of quantitative TLC. References :

1. VOVK, Irena, FRANKO, Mladen, GIBKES, Jύrgen, PROSEK, Mirko, BICANIC, Dane. Photoacoustic investigations of secondary chromatographic effects on TLC plates: [presented at] the fourth Asian Conference on Analytical Sciences, [also] ASIANALYSIS IV, Fukuoka University, Fukuoka, Japan, May 21-23, 1997. Anal . Sci . , 1997, vol. 13, suppl . , p. 191-194.

2. GIBKES, Jurgen, VOVK, Irena, BOLTE, Jochen, BICANIC, Dane, BEIN, Bruno, FRANKO, Mladen. Photother al characterization of thin-layer chromatography plates: presented at the Symposium on Achievements in Chromatography, Opatija, October 8-10, 1996. J. Chromatogr. , 1997, vol. 786, p. 163-170.

3. VOVK, Irena, FRANKO, Mladen, GIBKES, Jurgen, PROSEK, Mirko, BICANIC, Dane. Depth profiling of TLC plates by photoacoustic spectroscopy. JPC, J. Planar Chromatogr. Mod. TLC, 1997, 10, p. 258-262.

4. VOVK, Irena, FRANKO, Mladen, GIBKES, Jϋrgen, PROSEK, Mirko, BICANIC, Dane. Investigation by photoacoustic spectroscopy of the effect of drying conditions on the in- depth-distribution of compounds on TLC plates [The effect of drying conditions on the in-depth-distribution of compounds on TLC plates investigated by photoacoustic spectroscopy] . JPC, J. Planar Chromatogr . Mod. TLC, Sept/Oct 1998, vol. 11, no. 5, p. 379-382.

5. VOVK, Irena, GIBKES, Jurgen, FRANKO, Mladen, BICANIC, Dane. Depth profiling of TLC plates by photoacoustic spectroscopy. In: Proceedings of the 9th International Symposium, Interlaken, Switzerland, 9-11 April, 1997. Instrumental planar chromatography, p. 353-363 (L.22). 6. VOVK, Irena, FRANKO, Mladen, GIBKES, Jurgen, PROSEK, Mirko, BICANIC, Dane. The effect of drying conditions on the in-depth distribution of compounds on TLC plates investigated by photoacoustic spectroscopy. In: Inco COPERNICUS : minutes of the second meeting, Ebernburg, 26- 28 March, 1998, 12 p.

7. VOVK, Irena, PROSEK, Mirko. Iceberg in quantitative TLC. In: 40 years planar chromatography In Finland : abstracts [of symposium held in] Helsinki-Stockholm-Helsinki, 22-24 May, 1999 , Mis Silja Serenade, 1 p.

8. MOENIK, Grisa, VOVK, Irena, FRANKO, Mladen, GARDETTE, Gerard, KRAPEZ, Jean-Claude. Photothermal radiometric investigations of thin-layer chromatography plates. In: VOVK, Irena (ed. ) , PROSEK, Mirko (ed.), MEDJA, Armin (ed.). 1st International Meeting on Imaging Techniques in Planar Chromatography. Jezersko, Slovenia, May 14-16, 1999. Book of abstracts . [Ljubljana]: Slovenian Chemical Society: National Institute of Chemistry, 1999, p. 53-57.

9. VOVK, Irena, FRANKO, Mladen, GIBKES, Jurgen, PROSEK, Mirko, BICANIC, Dane. Photoacoustic investigations of secondary chromatographic effects on TLC plates. In: the Fourth Asian Conference on Analytical Sciences, Fukuoka, Japan, May 21-23, 1997. ASIANANALYSIS IV : abstracts , p. 34 (IA05) .

10. MOENIK, Grisa, VOVK, Irena, FRANKO, Mladen, OFFERMANN, Stephan, GIBKES, J rgen, GARDETTE, Gerard, KRAPEZ, Jean- Claude. Photothermal investigations of thin-layer chromatography plates. In: ORAEVSKY, Alexander A. (ed.). Biomedical opt o acoustics : 25-27 January 2000, San Jose, USA, (Progress in biomedical optics and imaging, vol. 1, no. 10), (SPIE proceedings series, vol. 3916). Bellingha , Wash., USA: SPIE - The International Society for Optical Engineering, cop. 2000, p. 171-178.

11. GIBKES, Jurgen, VOVK, Irena, BOLTE, Jochen, BICANIC, Dane, BEIN, Bruno, FRANKO, Mladen. Photothermal characterization of TLC plates. In: SEGUDOVIJI, Nikola. New- achievements in chromatography: book of abstracts international symposium : Opatija - Croatia, October 8- 10th, 1996. Opatija: Croatian Society of Chemical Engineers: Croatian Chemical Society, 1996, p. 62.

12. VOVK, Irena, PROSEK, Mirko. The effect of secondary chromatography on quantitative TLC. In: Proceedings of the Fifth Asian Conference on Analytical Sciences (ASIANANALYSIS V) : May 4-7, 1999 , Xiamen University, Xiamen, China , p. 287.

13. MOENIK, Grisa, VOVK, Irena, FRANKO, Mladen, OFFERMANN, Stephan, GIBKES, Jurgen, GARDETTE, Gerard, KRAPEZ, Jean- Claude. Photothermal investigations of thin-layer chromatography plates. In: BIOS 2000 - Biomedical optics, International biomedical optics symposium: technical abstract digest : 25-27 January 2000, San Jose, USA, p. 116-117 (3916-22).

14. VOVK, Irena, GIBKES, Jurgen, FRANKO, Mladen, BICANIC, Dane. Investigations of in-depth distribution of compounds on TLC plates by photoacoustic spectroscopy = Raziskave globinskih porazdelitev spojin na TLC ploseah z optoakustieno spektroskopijo. In: 10th International Symposium Spectroscopy in Theory and Practice, Bled, Slovenia, 14-17 April 1997. Book of abstracts . [Ljubljana] Slovenian Chemical Society, Spectroscopic Section, [1997], p. 189-190 (P-42). 15. VOVK, Irena, FRANKO, Mladen, GIBKES, Jurgen, PROSEK, Mirko, BICANIC, Dane. Photoacoustic investigations of effect of drying process on the in-depth distribution of compounds on TLC plates. In: Balaton Symposium on High- Performance Separation Methods, September 3-5, 1997, Siόfok, Hungary. Book of abstracts . [S. 1.]: [s. n.], 1997, p. 271 (P-151).

16. VOVK, Irena, FRANKO, Mladen, GIBKES, Jurgen, PROSEK, Mirko, BICANIC, Dane. The effect of drying conditions on the in-depth distribution of compounds on TLC and HPTLC plates investigated by photoacoustic spectroscopy. In: Euroanalysis X, September 6-11, 1998, Basel, Switzerland. Book of abstracts : Working Party on Analytical Chemistry, (Chimia, Vol. 52 (1998), no. 7/8), p. 347 (M26).

17. VOVK, Irena, FRANKO, Mladen, PROSEK, Mirko, BICANIC, Dane. Photoacoustic study of the in-depth distribution of compounds on TLC plates affected by drying conditions. In: 10th International Conference on Photoacoustic and Photothermal Phenomena, Rome, 23-27 August 1998. Book of abstracts . Roma: Aracne Editrice, 1998, p. 549.

18. VOVK, Irena, FRANKO, Mladen, GIBKES, J., PROSEK, Mirko, BICANIC, Dane. Photoacoustic study of the in-depth distribution of compounds on TLC and HPTLC plates affected by secondary chromatography. In: 5th. International Symposium Chromatography & Hyphenated Techniques, Bled (Slovenia), October 5-9, 1998. Book of abstracts . Ljubljana: Slovenian Chemical Society, 1998, p. 133.

19. VOVK, Irena, FRANKO, Mladen, GIBKES, Jurgen, PROSEK, Mirko, BICANIC, Dane. The effect of drying conditions on the in-depth distribution of compounds on TLC plates investigated by photoacoustic spectroscopy. In: Instrumental planar chromatography: proceedings of the 10th International Symposium, Visegrad, Hungary, 16-19 May, 1998. Budakalasz, Hungary: Research Institute for medicinal plants, 1998, p. 402-410.

20. VOVK, Irena, FRANKO, Mladen, GIBKES, Jurgen, PROSEK, Mirko, BICANIC, Dane. Photoacoustic study of the in-depth distribution of compounds on TLC plates affected by secondary chromatography. In: Oji international seminar on photothermal phenomena and their applications (ISPPA) : program [with abstracts] : New Oji , Hokkaido, August 5-7, 1998 , p. 255-258 (P22).

21. MOENIK, Grisa, VOVK, Irena, FRANKO, Mladen, OFFERMANN, Stephan, GARDETTE, Gerard, KRAPEZ, Jean-Claude. Photothermal investigations of thin-layer chromatography plates. In: Balaton symposium ' 99 on high-performance separation methods, September 1 -3 , 1999 , Siόfok (Hungary) : Book of abstracts .

21. MOENIK, Grisa, VOVK, Irena, FRANKO, Mladen, OFFERMANN, Stephan, GARDETTE, Gerard, KRAPEZ, Jean-Claude. Photothermal investigations of thin-layer chromatography plates. In: Balaton symposium ' 99 on high-performance separation methods, September 1 -3 , 1999 , Siόfok (Hungary) : Book of abstracts . Siόfok: Research Institute for Medicinal Plants, 1999 p. (P-57).

22. VOVK, Irena, PROSEK, Mirko. In-depth distribution in quantitative TLC. In: ADLARD, E. R. (ed.), COOKE, Michael (ed.), POOLE, C. F. (ed.), WILSON, Ian D. (ed. ) .

Encyclopedia of separation science . San Diego, Calif.; London: Academic Press, cop. 2000, vol. 7, p. 3087-3094.

23. VOVK, Irena. Inhomogeneous distribution of substances in the sorbent of the chromatographic plate and their influences on quantitative evaluation of chromatograms : doctoral thesis. (Nehomogene porazdelitve spojin v sorbentu kromatografske plosee in njihovi vplivi na kvantitativno vrednotenj e kromatogramov : doktorska disertacija) .

Ljubljana: [I. Vovk] , 1998. XII, 110 f., illustr. , tables. 24. VOVK, Irena, PROSEK, Mirko. Secondary chromatography and quantitative TLC: [presented at the] International Symposium on Instrumental ized Analytical Chemistry and Computer Technology (InCom ' 99) , Dϋsseldorf, 22-25 March 1999. Dϋsseldorf: Heinrich Heine Universitat, March 24, 1999.

Description of the solution of the technical problem with embodiments

The object of the invention is a device for controlled drying and derivatization in planar chromatography by directed air or inert-gas flow.

The invention is based on our findings that the conspicuous dispersion of results in quantitative thin-layer chromatography (TLC) is due to uncontrolled drying of developed chromatographic plates and to uncontrolled derivatization conditions. As mentioned earlier, in uncontrolled drying a secondary chromatography occurs, leading, in turn, to a nonhomogeneous distribution of molecules of the sample inside the sorbent layer and consequently to different absorption and light scattering during measurement. Under uncontrolled derivatization conditions, reactions tend to take place in a nonuniform manner, with additional unwanted effects on the accuracy and the reproducibility of the analyses .

In order to overcome the problems discussed above we have developed a device, shown in accompanying Figure 1, by means of which the problem of drying chromatographic plates, as well as the problem of derivatization, are solved integrally. Said device overcomes the disadvantages of prior-art devices and provides fast, controlled, directed and reproducible drying of chromatographic plates and derivatization under homogeneous conditions at controlled temperature and in an arbitrarily directed air or inert-gas flow, additionally enabling integration into a complete and automated system of chromatogram management and evaluation (e.g. drying - derivatization - densitometry - video documentation - calculations).

The main characteristics of the inventive device are as follows : the heated air flow is directed over the sorbent layer in the direction of chromatogram development. The temperature of the heated air is preferably not constant but regularly oscillating between a minimum and a maximum value. The range between said temperature values, as well as the length of the oscillation interval, may be regulated using a dedicated program. The device also supports computerized drying and automated operation through integration into a complete system for the processing and evaluation of chromatograms and results.

The accompanying Figure 1 shows the device according to the invention, consisting of a housing 1 with a glass cover 2 to monitor drying (referring to the derivatization option), a drawer 3 for accommodating TLC plates, a heater 4, a fan 5, a temperature sensor 6, an exhaust opening 7 for expelling air (into the digester), an inlet opening 8, fitted with a filter, for supplying air or inert gas, a shutter 9 for obtaining laminar flow, a fan operation controller 10, a heater control unit 11, a timer unit 12 with general controller, and an interface 13 for connecting external control units (RS232).

The main characteristics of the inventive device are as follows :

• It provides programmed drying of chromatographic plates and derivatization of chromatograms under controlled conditions .

• Heat is supplied in regular or variable time intervals .

• The temperature gradient in the direction of the air flow is constant, and temperature is uniform over the entire plate width.

• Heating grade, heating intervals, drying time (switching on and off) and other parameters may be selected in advance, or controlled via a computer program.

• Drying is achieved with a directed laminar air flow.

Consequently, the following advantages are achieved: • Laminar air flow constantly removes solvent vapors from the upper layers of the sorbent, thereby accelerating drying; hence, diffusion of analyte components and band broadening are prevented; • Controlled temperature variation prevents over- concentration of the mobile phase in the upper layers of the sorbent.

• Heated air accelerates drying.

• Due to the directional air flow, band broadening caused by drying, if at all present under given conditions, is not random but controlled in the flow direction; in addition, homogeneity of the temperature gradient as well as temperature uniformity over the entire plate width is achieved, further reducing potential deficiencies imputable to nonhomogeneous drying.

• Overheating and consequent degradation of analyte components is avoided through temperature control.

• By programming and controlling the drying parameters, predefined drying conditions may be achieved in a reproducible and controlled manner, improving the reproducibility of the in-depth spots ' position and shape.

• Controlled conditions improve the accuracy and the reproducibility of the derivatization process.

• Drying and derivatization may be incorporated into an integral system of controlled chromatogram management and evaluation.

As a consequence of said advantages of the inventive device over conventional devices for drying chromatographic plates, the errors and the dispersion of results in thin- layer chromatography analyses due to drying and derivatization are minimized, whereas the accuracy and the reproducibility of such analyses are significantly improved, as illustrated by the comparison of results listed in Table 1 and in Diagrams 1 and 2 below.

In addition, the drying device of the invention allows the number of parallel analyses of the analyte to be reduced without increasing the error of the analysis.

Thus the device of the invention allows to lower the costs of thin-layer chromatography analysis while greatly improving the utility and the reliability thereof, which is important both for economical reasons and for ensuring quality standards .

The invention is illustrated by the following embodiments, which are not limiting as to its scope.

First embodiment: Device for drying chromatographic plates

The device is formed as shown in Figure 1. The housing is made of plastic or metallic material and has an inlet opening for introducing air or inert gas from the environment and an exhaust opening for expelling air or inert gas from the system. The flow of the fluid is ensured by means of a fan of constant or variable speed and is directed by a shutter or a baffle plate.

Air is heated by a 100-2000 W rating multistage heater, the minimum and maximum values of the drying temperature being regulated through selection of the heating grade (power) and the on/off switching frequency thereof. The heater on/off switching frequency may be preset by means of the incorporated timer unit whereby a suitable on/off switching frequency may be selected, or via a computer program (Figure 2 - diagram of the interdependence between temperature and the selected heating/cooling program) .

The chromatographic plate is inserted into the device using a retractable drying drawer, whereon the plate is placed facing any convenient direction, e.g. in such a way that the air flow is directed in the direction of chromatogram development. The device may optionally be provided with a system for recording all data about the drying process in progress .

Second embodiment: Device for drying chromatographic plates and derivatization with a transparent cover

The device is similar to the first embodiment, differing therefrom in that the housing over the drying ' drawer is transparent, for example made of glass.

Third embodiment: Automated device for drying chromatographic plates and derivatization The device is similar to the first and second embodiments, and is enhanced with a system of pre-selectable and/or programmable drying.

Fourth embodiment: Utilization of the inventive device to dry chromatographic plates - spot application

6 chromatographic plates, each with 18 applications 10 mm from each other were prepared. The analyte was dye mixture Camag test dye III (Camag, Muttenz, Switzerland). The amount of the applications was 1 ig per spot. The chromatographic plates were developed in toluene under identical conditions . Two plates were dried with a hair dryer (1000 W, 5 in, switched on and off in regular 30- second time intervals, air flowing in the direction of chromatogram development), two with a heating plate (Camag, Muttenz, Switzerland; 5 min, 80°C) and two with the drying device of the invention as described in the first embodiment. Thereafter, the chromatograms were recorded with the Camag TLC II densitometer in remission and in transmission. For each application giving five visible bands, the area and the height of three characteristic peaks with different Rf values were measured and evaluated. Thus 108 measurements were taken for each plate, 216 measurements for each type of drying device, amounting to 648 measurements in all. The results are listed in Table 1, the comparison being also shown in the diagrams of Figures 3 and 4. Table 1. Dispersion of results obtained by drying with the inventive device (TLC dryer), a hair dryer and a heating plate, respectively, expressed by relative standard deviation (RSD). 216 measurements were taken for each type of drying device.

The accompanying chart of Figure 3 shows comparative RSD values for the measurements of the area of the bands obtained by drying chromatograms with a heating plate, with the inventive TLC dryer, and with a hair dryer, respectively.

The accompanying chart of Figure 4 shows comparative RSD values for the measurements of the height of the peaks obtained by drying chromatograms with a heating plate, with the inventive TLC dryer, and with a hair dryer, respectively.

Fourth embodiment : Measurement of the surface temperature of the chromatographic plate in the course of programmed drying

The chromatographic plate was dried in the inventive TLC dryer using a program with equal heating and cooling time intervals . The temperature was measured on the surface of the chromatographic plate. The resulting temperature profile may be seen in Figure 5 (T measured in °C, time measured in seconds ) .

Claims

Patent Claims

1. Device for controlled drying and derivatization in planar chromatography, characterized in that controlled drying and derivatization is achieved by directed, heated or non-heated air or inert gas .

2. Device for controlled drying and derivatization in planar chromatography according to Claim 1 , characterized in that it consists of a housing (1) with an optional transparent cover (2) to monitor drying, a drawer (3 ) for accommodating TLC plates, a heater ( 4 ) , a fan (5), a temperature sensor (6), an exhaust opening (7) for expelling air, an inlet opening (8), fitted with a filter, for supplying air or inert gas, a shutter (9) for obtaining laminar flow, a fan operation controller (10), a heater control unit (11), a timer unit (12) with general controller, and an interface (13) for connecting external control units (RS232).

3. Device as claimed in any one of the preceding claims, characterized in that the cover above the drying compartment is made of glass.

4. Device as claimed in any one of the preceding claims, characterized in that its operation is programmed or preset.

5. Device as claimed in any one of the preceding claims, characterized in that its operation is automated.

6. Device as claimed in any one of the preceding claims, characterized in that it is operated through the use of a computer.

7. Device as claimed in any one of the preceding claims, characterized in that it is integrated into a complete system for processing and evaluating chromatograms and results.

8. Usage of a device as claimed in any one of the preceding claims, characterized in that it enables drying and derivatization in a directed laminar flow of air or inert gas at a controlled constant or variable temperature, preferably at a regularly oscillating temperature.

9. Usage of a device as claimed in any one of the preceding claims, characterized in that it enables drying and derivatization at temperatures between room temperature and 200°C.

10. Usage of a device as claimed in any one of the preceding claims, characterized in that the chromatographic plate is dried under predefined conditions .

11. Usage of a device as claimed in any one of the preceding claims, characterized in that drying occurs in an air or an inert-gas flow which is directed so as to flow in the direction of chromatogram development or in the inverse direction relative to chromatogram development.

12. Usage of device as claimed in Claim 4, characterized in that derivatization is performed under controlled conditions with the possibility of video recording.