SI21199A - Device for controlled drying and derivatization in chromatography on plates and its use - Google Patents

Abstract

A device enables an essentially improved repeatability of measurements in quantitative thin layer chromatography TLC and HPTLC. Better correctness, sensitivity and repeatability is achieved by prevention of uncontrolled spreading of spots due to diffusion, ununiform vertical distribution of spots in sorbent layer during drying as well as inhomogeneous conditions during derivatization. The basic properties of the device developed are: the flow of heated air is directed over the sorbent layer in such a manner that it follows the direction of chromatogram development. Temperature of heated air is not constant preferentially, but oscillates uniformly between the lower and the upper values of temperature. The span between the upper and the lower temperature values as well as the oscillation period is varied according to a special program. The device enables also the computer control of drying and automation by inclusion into an integral system of processing and evaluation of chromatograms or results respectively.

Description

Direction of drawer movement

-1 Controlled drying and derivatization apparatus for plate chromatography and its use

The field of technology

The invention relates to the field of analytical chemistry and relates to a device for controlled drying and derivatization in plate chromatography and to its use for providing reproducible and controlled execution of individual phases of thin layer chromatography. Better regularity, sensitivity and reproducibility are achieved by preventing the uncontrolled spread of stains due to diffusion, the uneven vertical distribution of stains in the sorbent layer during drying and homogeneous derivatization conditions.

A technical problem

When performing quantitative thin-layer chromatography (TLC) analyzes, one of the key problems is the proper drying of the developed chromatographic plates, since many of the analysis parameters, such as e.g. the sensitivity, regularity and repeatability of the results depend significantly on this phase. Therefore, there is a need for a device that allows controlled and technically appropriate drying and other operations to develop chromatograms (eg derivatization).

The state of the art

In the world, drying of chromatographic plates is not given special attention because it is unjustifiably considered that drying of the plates is not a great source of error. Manufacturers are developing devices for the application and development of chromatograms, and leave the decision on how to dry and derivatize to laboratories.

For this reason, conventional laboratory dryers and hair-dryer-type dry hair dryers ("hair dryers") are not used in the laboratories for proper drying. Thus, in the case of the use of the heating plate, it is uneven and therefore causes an uneven distribution of stains in the layer, but may also cause overheating and degradation of individual components of the analyte. In the laboratory dryer, there is a temperature gradient, so the temperature is not the same throughout the TLC panel, which results in uneven drying, except that laminar air flow cannot be achieved and channeled across the panel in the same direction as they were developed. uncontrolled spreading of stains in different directions.

Because drying with a hair dryer or. poorly reproducible in the laboratory dryer and often the cause of incorrect or poorly reproducible analysis results, various manufacturers tried to solve the drying problem by developing special heating plates with integrated temperature control. These plates are similar in appearance to electric cookers and are designed so that the temperature on the surface of the hob is as uniform as possible. However, the heating plate only supplies energy and does not remove the vapors of the resulting mobile phase. Thus, the solvent vapor accumulates on the surface of the sorbent, resulting in a horizontal spread of stains and, consequently, defects or defects. uniqueness of results. However, errors can sometimes be the result of accidental vapor removal due to the uncontrolled flow of air in the workspace. in the vicinity of the chromatographic plate. As a result, repeatability or the correctness of the results is not satisfactory even when using the heating plates.

In the case of derivatization, the weakness of the heating plates is mainly reflected in the uneven temperature across the surface of the plate (sorbent), especially due to the uncontrolled heat transfer to the surroundings. Therefore, there are differences in derivatization reactions on different parts of the plate and consequently poorer regularity and repeatability.

The problem of large scattering of results is, in practice, solved by applying multiple parallels of the same sample and standard to the plate and using a larger number of chromatographic plates, ie by averaging from a larger number of results. All of this significantly reduces the usefulness of quantitative TLC.

References:

1. VOVK, Irena, FRANKO, Mladen, GIBKES, Jurgen, PROŠEK, 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. [COBISS-ID 12807975]

2. GIBKES, Jurgen, VOVK, Irena, BOLTE, Jochen, BICANIC, Dane, BEIN, Bruno, FRANKO, Mladen. Photothermal 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. [COBISS-ID 12737319]

3. VOVK, Irena, FRANKO, Mladen, GIBKES, Jurgen, PROŠEK, Mirko, BICANIC, Dane. Depth profiling of TLC plates by photoacoustic spectroscopy. JPC, J. planar chromatogr. mod. TLC, 1997, 10, p. 258-262. [COBISS-ID 3984679]

4. VOVK, Irena, FRANKO, Mladen, GIBKES, Jurgen, PROŠEK, 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. [COBISS-ID 11515]

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). [COBISS-ID 1537562]

6. VOVK, Irena, FRANKO, Mladen, GIBKES, Jurgen, PROŠEK, Mirko, BICANIC, Dane. The effect of drying conditions on the in-depth distribution of compounds on TLC plates investigated by photoacoustic spectroscopy. In: Incho COPERNICUS: Minutes of the Second Meeting, Ebernburg, March 26-28, 1998, 12 p. [COBISS-ID 1748250]

7. VOVK, Irena, PROŠEK, Mirko. Iceberg and quantitative TLC. V. 40 years planar chromatography in Finland: abstracts [of symposium held in] Helsinki-StockholmHelsinki, May 22-24, 1999, M / s Silja Serenade, 1 p. [COBISS-ID 1906458]

8. MOČNIK, Grisa, VOVK, Irena, FRANKO, Mladen, GARDETTE, Gerard, KRAPEZ, Jean-Claude. Photothermal radiometric investigations of thin-layer chromatography plates. In: VOVK, Irena (ed), PROŠEK, Mirko (ed), MEDJA, Armin (ed). 4th 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. [COBISS-ID 1897242]

9. VOVK, Irena, FRANKO, Mladen, GIBKES, JCirgen, PROŠEK, 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). [COBISS-ID 1531162]

10. MOČNIK, Grisa, VOVK, Irena, FRANKO, Mladen, OFFERMANN, Stephan, GIBKES, Jurgen, GARDETTE, Gerard, KRAPEŽ, Jean-Claude. Photothermal investigations of thin-layer chromatography plates. In: ORAEVSKY, Alexander A., editor (s). Biomedical optoacoustics: 25-27 January 2000, San Jose, USA, (SPIE proceedings series, vol. 3916) (Progress in biomedical optics and imaging, vol. 1, no. 10). Bellingham, Wash., USA: SPIE-The International Society for Optical Engineering, cop. 2000, p. 171-178. [COBISS-ID 2148378]

11. GIBKES, JCirgen, VOVK, Irena, BOLTE, Jochen, BICANIC, Dane, BEIN, Bruno, FRANKO, Mladen. Photothermal characterization of TLC plates. In: SEGUDOVIC, 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. [COBISS-ID 749594]

11. VOVK, Irena, PROŠEK, 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. [COBISS-ID 1907226]

13. MOČNIK, Grisa, VOVK, Irena, FRANKO, Mladen, OFFERMANN, Stephan, GIBKES, Jurgen, GARDETTE, Gerard, KRAPEŽ, Jean-Claude. Photothermal investigations of thin-layer chromatography plates. In: BIOS 2000 - Biomedical optics, International biomedical optics symposium: technica! abstract digest: 25-27 January / 2000, San Jose, USA, p. 116-117 (3916-22). [COBISS-ID 2150938]

14. VOVK, Irena, GIBKES, Jurgen, FRANKO, Mladen, BICANIC, Dane. Investigations of in-depth distribution of compounds on TLC plates by photoacoustic spectroscopy = Investigation of depth distributions of compounds on TLC plates by optoacoustic spectroscopy. 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). [COBISS-ID 3836199]

15. VOVK, Irena, FRANKO, Mladen, GIBKES, Jurgen, PROŠEK, Mirko, BICANIC, Dane. Photoacoustic investigations of the effect of drying process on the in-depth distribution of compounds on TLC plates. In: Balaton Symposium on HighPerformance Separation Methods, September 3-5, 1997, Siofok, Hungary. Book of abstracts. [S. I.]: [s. n.], 1997, p. 271 (P-151). [COBISS-ID 1532954]

16. VOVK, Irena, FRANKO, Mladen, GIBKES, Jurgen, PROŠEK, 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: Vorking Party on

Analytical Chemistry, (Chimia, Vol. 52 (1998), No. 7/8), p. 347 (M26). [COBISS-ID 1764890]

17. VOVK, Irena, FRANKO, Mladen, PROŠEK, 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. [COBISS-ID 13407271]

18. VOVK, Irena, FRANKO, Mladen, GIBKES, J., PROŠEK, 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. [COBISSID 13468455]

19. VOVK, Irena, FRANKO, Mladen, GIBKES, Jurgen, PROŠEK, Mirko, BICANIC, Dane. The effect of drying conditions on the in-depth distribution of compounds on TLC plates investigated by photoacoustic spectroscopy. In: Instrumenta / planar chromatography: proceedings of the 10th International Symposium, Visegrad, Hungary, May 16-19, 1998. Budakalasz, Hungary: Research Institute for Medicinal Plants, 1998, p. 402-410. [COBISS-ID 1747738]

20. VOVK, Irena, FRANKO, Mladen, GIBKES, Jurgen, PROŠEK, 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). [COBISS-ID 1884954]

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

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

Siofok: Research Institute for Medicinal Plants, 1999, 1 p. (P-57). [COBISS-ID 2038042]

21. VOVK, Irena, PROŠEK, Mirko. In-depth distribution and quantitative TLC. In: ADLARD, E. R. (ed.), COOKE, Michael (ed.), POOLE, C. F. (ed.), VVILSON, lan D. (ed.). Encyclopedia of separation Science. San Diego, Calif .; London: Academic Press, cop. 2000, vol. 7, p. 3087-3094. [COBISS-ID 2203418]

22. VOVK, Irena. Inhomogeneous distributions of compounds in the chromatographic plate sorbent and their effects on the quantitative evaluation of chromatograms: doctoral thesis. Ljubljana: [I. Vovk], 1998. XII, 110 f., Illust., Tables. [COBISS-ID 42794497]

23. VOVK, Irena, PROŠEK, Mirko. Secondary chromatography and quantitative TLC: [presented at the International Symposium on Instrumentalized Analytical Chemistry and Computer Technology (lnCom'99), Dusseldorf, March 22-25, 1999. Dusseldorf: Heinrich Heine University, March 24, 1999. [COBISS-ID 1845274]

Description of solution to a technical problem with implementation examples

The subject of the invention is a device (dryer) for controlled drying and derivatization in chromatography on directional airflow plates. of inert gas.

The basis of the invention is our finding that the large scattering of results in quantitative thin layer chromatography (TLC) is due to the uncontrolled drying of the developed chromatographic plates and the uncontrolled derivatization conditions. As mentioned earlier, uncontrolled drying results in secondary chromatography, which affects the uneven distribution of the sample molecules in the sorbent layer and thus different absorption and scattering of light during measurement. Uncontrolled derivatization conditions result in an uneven flow of reactions, which additionally affects the accuracy and repeatability of the analyzes.

In order to solve these problems, we have developed the device shown in the accompanying Figure 1, which solved the problem of drying the chromatographic plates integrally, as well as the problem of derivatization. The device eliminates the disadvantages of the existing devices, enables fast, controlled, controlled and repeatable drying of the chromatographic plates and derivatization under homogeneous conditions at a controlled temperature in the chosen direction of air flow or. of inert gas, but also enables integration into the system of complete and automated preparation and evaluation of chromatograms (eg drying-derivatization-densitometry and video-documenting-calculation).

The basic features of the developed device are: the heated air stream is directed through the sorbent layer so that it flows in the direction of developing the chromatograms. The temperature of the heated air is preferably not constant but fluctuates uniformly between the lower value and the upper temperature value. The range of the lower and upper temperature values, as well as the oscillation time, is varied with a special program. The device also enables computerized control of drying and automation by including them in a comprehensive system for the processing and evaluation of chromatograms. results.

The accompanying Figure 1 shows a device according to the present invention consisting of a housing 1 with a glass lid 2 for controlling the drying (with a derivatization option), a drawer 3 for installing a TLC board, a heater 4, a fan 5, a temperature meter 6, a drainage opening 7 air (in the digestion room), openings 8, fitted with a filter, for supplying air or air. inert gas, apertures 9 to achieve laminar flow, units 10 for controlling and controlling fan operation, units 11 for controlling and controlling the operation of the heater, units 12 for timing and linking controls, and an interface 13 for connecting to external control units (RS232).

The main features of the device are:

• allows programmed drying of chromatographic plates and derivatization of chromatograms under controlled conditions • heat supply occurs at regular or variable intervals • temperature gradient in the direction of air flow is constant while temperature across the entire width of the plate is equal • heating rates, heating intervals, drying time (on and off) and other parameters are adjustable or adjustable. guided by a computer-driven program • drying takes place with a laminar directional flow of air.

In this way we achieve the following advantages:

• With laminar air flow, solvent vapors are continuously removed from the upper sorbent layers and accelerated drying; this prevents the diffusion of the analyte components, spreading stains;

• By controlling the temperature fluctuation, the concentration of the mobile phase in the upper layers of the sorbent is further prevented;

• By heating the air we accelerate drying;

• By directing the air flow, the expansion of the spots due to drying, if it occurs in the given conditions, is made to be non-random, controlled in the direction of the air flow; at the same time, we achieve the homogeneity of the temperature gradient and the uniformity of temperature over the entire width of the plate, thereby further reducing errors due to uneven drying;

• Temperature control prevents overheating and decay of analyte components;

-1010 • By programming and controlling the drying parameters, the desired drying conditions are achieved in a repeatable and controlled manner, thus affecting the reproducibility of the position and shape of the spots;

• By controlling the conditions, we increase the regularity and repeatability of derivatization. • Drying and derivatization can be integrated into an integrated system of guided preparation and evaluation of chromatograms.

The aforementioned advantages of the present device over other devices for drying the chromatographic plates are the minimization of errors and scattering of the results of thin-layer chromatography analyzes for drying and derivatization and a significant increase in the accuracy and repeatability of these analyzes, as illustrated by the comparative results in Table 1 and Diagrams 1 and 2.

At the same time, the use of a subject drying apparatus allows the number of parallel analyzes of the analyte to be reduced without increasing the error of analysis.

Thus, the device in question reduces the cost of analysis by thin layer chromatography and substantially increases its usefulness and reliability, which is important from the point of view of economy and quality standards.

The invention is illustrated but not limited by the embodiments.

Example 1: A device for drying chromatographic plates

The device is assembled as shown in Figure 1. The housing is plastic or metal, with openings for air or inert gas supply from the surroundings and air or air outlet. of inert gas from the system. The flow of gas is provided by a fan with a constant or adjustable speed, which is directed by an aperture or. routers.

For the air heating we use a multi-stage heater with a power of 100-2000 W, the lower and upper drying temperatures are regulated by the choice of heating rate (power) and the switching frequency or. switch off the heating.

-11The heating on / off frequency is set with a built-in time controller with the option of setting the on / off frequency or using a computer-controlled program (Figure 2 - showing the relationship between temperature and the selected heating and cooling program).

The chromatographic plate is inserted into the device via a pull-out desiccant, on which the plate is placed in any direction, e.g. so that the flow of air is directed in the direction of developing the chromatogram. The device may also be equipped, if necessary, with a system for recording all drying progress data.

Example 2: A device for drying chromatographic plates and derivatization with a transparent lid

The apparatus is assembled as in Example 1 with the housing above the drying table being transparent, for example glass.

Example 3: An automated device for drying chromatographic plates and derivatization

The device, as described in Examples 1 and 2, is supplemented by a preset system. drying programming.

Example 4: Use of the device of the invention for drying the chromatographic plates - application in point

We prepared 6 chromatographic plates with 18 coats each, at a point with a coating distance of 10 mm. The analyte was a dye mixture of Camag test dye III (Camag, Muttenz, CH). The coating size was 1 pg / coat. Chromatographic plates were developed under the same conditions in toluene. Two plates were each dried with a blow dryer (1000 W, 5 min, switching on and off at equal intervals of 30 s, air flow in the direction of chromatogram development), drying plate (Camag, Muttenz, CH; 5 min, 80 ° C) and subject the drying apparatus described in Example 1. The chromatograms were then taken with Camag's TLC II

-1212 densitometer in remission and transmission. In each application, the areas and heights of three characteristic spots with different Rf values were measured and evaluated from the five visible spots. Thus, from each panel, 108 measurements were processed for 216 measurements for each drying method, totaling 648 measurements in the experiment. The results are shown in Table 1 and a comparison is also made in the diagrams in Figures 3 and 4.

Table 1. Scattering of the results, expressed in relative standard deviation (RSD) obtained by drying with the subject drying apparatus (Dryer), hair dryer and drying plate. There are 216 measurements for each type of drying.

Remission transmission Drying type height Surface height surface Dryer ± 2.75% ± 3.75% ± 2.39% ± 3.37% Hair dryer ± 4.19% ± 5.57% ± 3.92% ± 5.57% Heating plate ± 4.61% ± 6.20% ± 4.60% ± 6.50%

The accompanying diagram in Figure 3 shows the comparison of RSD in measurements of the spots of the spots after drying the chromatograms with a heating plate, a subject dryer and a hair dryer.

The accompanying diagram in Figure 4 shows the comparison of RSD in measurements of the height of spots after drying the chromatograms with a heating plate, a subject dryer and a hair dryer.

Example 4: Measurement of temperature on the surface of a chromatographic plate while performing programmed drying

-1313

The chromatographic plate was dried in a subject dryer using a program with the same heating and cooling intervals. The temperature was measured on the surface of the chromatographic plate. The temperature profile shown in Figure 5 (T in ° C, time in seconds) was obtained.

Claims (12)

1. Controlled drying and derivatization device in plate chromatography, characterized in that the controlled drying and derivatization in plate chromatography is realized with directional, heated or unheated, air or inert gas. Controlled drying and derivatization device in plate chromatography according to claim 1, characterized in that it consists of a housing (1) with an optional transparent cover (2) for drying control, a drawer (3) for mounting a TLC plate, a heater (4 ), fan (5), temperature gauge (6), air outlet openings (7), openings (8) fitted with a filter for air or air inlet. inert gas, diaphragm (9) for laminar flow, unit (10) for controlling and controlling fan operation, unit (11) for controlling and controlling heater operation, unit (12) for timing and linking controls and interface (13) for connection to external control units (RS232). Apparatus according to the preceding claims, characterized in that the cover above the drying table is glass. Device according to the preceding claims, characterized in that its control is programmed. Device according to the preceding claims, characterized in that its operation is automated. Device according to the preceding claims, characterized in that it is controlled by a computer. -1515 Apparatus according to the preceding claims, characterized in that it is integrated into the whole system of processing and evaluation of the chromatograms. results. Use of an apparatus according to any one of the preceding claims, characterized in that it allows drying and derivatization with a directed flow of air or inert gas at a controlled constant or varying temperature, preferably at a uniformly fluctuating temperature. Use of a device according to one of the preceding claims, characterized in that it allows drying and derivatization at room temperatures up to 200 ° C. Use of an apparatus according to one of the preceding claims, characterized in that the chromatographic plate is dried under the selected conditions. Use of the device according to one of the preceding claims, characterized in that the drying takes place in the air stream or in the air. inert gas such that the air is directed in the direction of development of the chromatogram or in the opposite direction of development of the chromatogram. Use of a device according to claim 4, characterized in that derivatization is carried out under controlled conditions with video capability.