Evaluation method for thin-layer chromatography plates

Abstract

A method for evaluating a thin layer chromatography plate (2) after a separation process for separating components of a sample on the thin layer chromatography plate (2) includes a digitization step in which at least two digital images (1, 5) that differ in terms of the wavelength range of illumination of the thin layer chromatography plate (2) are taken. The method further includes a position identification step in which a position on the thin layer chromatography plate (2) is identified for each of the at least two digital images (1, 5). The method further includes an evaluation step in which image information from the at least two digital images (1, 5) is superimposed for at least all areas having at least one visible spot (9, 10) in at least one of the at least two digital images (1, 5), such that the superimposed image information (10) from the same area of ​​the at least two digital images (1, 5) can be used to evaluate the thin layer chromatography.

Description

Technical Field

[0001] The present invention relates to a method for evaluating a thin layer chromatography plate after performing a separation process for separating components of a sample on the thin layer chromatography plate, whereby, within a digitization step, a digital image of the thin layer chromatography plate is taken using a digital image device, whereby, within a position identification step, the position of the thin layer chromatography plate within the digital image is identified, and whereby, within an evaluation step, the position of visible spots indicating the sample components on the thin layer chromatography plate is determined, and the thin layer chromatography is evaluated based on the determined spot positions.

Background Art

[0002] Background Thin layer chromatography is a chromatography technique used to separate non-volatile mixtures within a sample, enabling the identification and, in some cases, quantification of the individual components of the sample. Thin layer chromatography is typically performed on a thin layer chromatography plate, which can be a glass or plastic plate, or aluminum foil, coated with a thin layer of an adsorbent material such as silica gel, aluminum oxide, or cellulose. This layer of adsorbent is known as the stationary phase. After the sample is applied onto the plate, a solvent or solvent mixture known as the mobile phase is drawn up onto the plate by capillary action. When the sample is applied onto a region near the boundary of the plate and the solvent mixture is drawn up from the same boundary of the plate, the sample is drawn up along with the solvent mixture across the plate. Since different components of the sample, i.e., different analytes, rise up the plate at different rates, separation of the analytes is achieved, and as a result, different spots are formed along the path of the solvent mixture that is drawn up and moves across the plate.

[0003] Following the development of the thin-layer chromatography plate, spots of different analytes in the sample become visible. This can be done by illuminating the plate with visible light or ultraviolet light. Chemical processes can also be used to improve the visibility of the spots. The position and size of the spots on the plate can be used for analyte identification and quantitative measurement of the amount of each analyte in the sample.

[0004] To identify components, the migration distance of the analyte under consideration is divided by the total migration distance of the mobile phase, i.e., the solvent mixture. This ratio is called the retardation factor. While the retardation factor is unique, it will vary depending on the exact conditions of the mobile and stationary phases. Therefore, typically, before developing the plate and analyzing the results, samples of known compounds and one or more target samples are combined on the plate. Thin-layer chromatography can be used to monitor the progress of a reaction, identify compounds present in a given mixture, and determine the purity of a substance. Thus, thin-layer chromatography is an established method for analyzing samples in many different application environments, such as research and development, as well as monitoring and quality control of industrial production processes.

[0005] There are several thin-layer chromatography plate evaluation devices that enable semi-automated or fully automated steps for evaluating thin-layer chromatography plates unfolded by a separation process. Some devices provide a digitization chamber with a tray or slot for receiving the thin-layer chromatography plate. The plate is then illuminated with a light source, and a digital image is taken using a digital imaging device, such as a digital camera or even a smartphone. At least some of the devices already available provide software that enables the visualization and identification of visible spots in such digital images taken from the thin-layer chromatography plate. Sometimes, the evaluation of digital images of thin-layer chromatography plates is performed using standalone software running on a computer. However, these known methods for evaluating digital images have several drawbacks and limitations. Typically, the software used to evaluate unfolded thin-layer chromatography plates must be adapted to the evaluation of plates of a given size, and the position and orientation of the plate must be clearly defined during the digitization step. Only then can the pixels of the digital image be referenced to their corresponding positions on the thin-layer chromatography plate. The use of such software is usually limited to one shape of thin-layer chromatography plate. The evaluation results depend heavily on the manually specified placement and orientation of the plate during digital image recording. Furthermore, it is well known that the identification and evaluation of visible spots on thin-layer chromatography plates are limited by the quality of the digital images taken from the thin-layer chromatography plates. In addition, the visibility of spots of a separated sample on the surface of a thin-layer chromatography plate also depends on the illumination of the plate during the digitization step, i.e., the wavelength or emission characteristics of the illumination device used to illuminate the plate. Therefore, there is a need for a method that allows more information, and preferably more accurate information, to be obtained from digital images taken from thin-layer chromatography plates after development. [Overview of the Initiative]

[0006] As described above, the present invention relates to a method for evaluating a thin-layer chromatography plate using a digitization step, a position identification step, and an evaluation step, wherein in the digitization step, at least two digital images different in terms of illumination wavelength range are captured, in the position identification step, the position of the thin-layer chromatography plate is identified for each of the at least two digital images, and in the evaluation step, the image information of at least two digital images is superimposed for at least all regions having at least one visible spot in at least one of the at least two digital images, and the superimposition of image information from the same region of at least two digital images can be used for the evaluation of thin-layer chromatography. The present invention takes advantage of the fact that different illumination of the same plate results in different visibility of spots of sample material separated on the surface of the thin-layer chromatography plate. Therefore, a spot in a region of the plate that is clearly visible under first illumination may be difficult to see or even difficult to identify under second illumination. However, by evaluating several digital images of the same plate captured using different illumination, it is possible to identify more spots and obtain more accurate results by analyzing more spots compared to the results that can be obtained from a single digital image.

[0007] It is advantageous to capture two or more different digital images of a thin-layer chromatography plate using different lighting without moving the thin-layer chromatography plate and the optical imaging device relative to each other. Therefore, the same pixel in each digital image corresponds to the same point on the surface of the thin-layer chromatography plate, and thus, for each digital image, the same pixel can be referenced with the same real-world coordinates corresponding to that pixel, without requiring optical image recognition and optical image analysis methods to identify the shape and position of the thin-layer chromatography plate within each digital image. By referencing pixels in a digital image with real-world coordinates that refer to a position on the surface of the thin-layer chromatography plate, the actual location of a spot of unfolded sample material on the thin-layer chromatography plate can be identified. Such information can be used for subsequent optical marking of spots in a visual reproduction of the thin-layer chromatography plate for user visual inspection, or for subsequent analysis of sample material taken from a selected spot at a given location on the surface of the thin-layer chromatography plate. Furthermore, by referencing pixels in a digital image with real-world coordinates, it becomes possible to identify the location of a spot in a digital image that is invisible or barely visible in this digital image, but is visible in other digital images taken using different lighting.

[0008] A single digital image may contain one or more thin-layer chromatography plates arranged adjacent to each other. For example, two or three thin-layer chromatography plates can be arranged in a row, or two, four, or six thin-layer chromatography plates can be arranged in a matrix, and all of these plates can be captured in a single digital image. It is even possible to arrange any number of thin-layer chromatography plates in any arrangement, in different directions and at different distances from each other, and capture all of these plates in a single digital image. The single digital image can then be automatically analyzed, and all thin-layer chromatography plates contained within the single digital image can be identified by utilizing known image processing software modules that identify the boundaries of each thin-layer chromatography plate and the regions adjacent to them. Subsequently, the evaluation method described above can be performed for each thin-layer chromatography plate individually, i.e., without referring to information based on or related to other thin-layer chromatography plates.

[0009] To enable a fully automated evaluation process, two digital images are taken from the same plate but under different lighting conditions. In a subsequent position identification step, the two digital images are analyzed using image analysis methods to identify the shape, position, and orientation of the plate within the digital images. Using well-known methods and readily available software modules, it is possible to automatically identify the shape, position, and orientation of the plate within the digital images. Thus, after the completion of the position identification step, each pixel in each digital image can be referenced to a position on or near the surface of the thin-layer chromatography plate. By performing such a fully automated position identification step, accurate positional information regarding the thin-layer chromatography plate associated with each digital image is provided without requiring manual input or manual image analysis. Furthermore, it is also possible to overlay the two digital images in a way that overlays correlated positions and regions of the thin-layer chromatography plate. Overlaying the digital images results in improved image information for each position on the surface of the thin-layer chromatography plate. If such image information overlay is performed for all regions containing a spot in at least one digital image, the overlay adds information related to the spot from both digital images. Even if a spot is visible to the human eye as only one part of a digital image, there may be small contributions from other digital images that are invisible to the human eye, but nevertheless enhance the superimposed image information, allowing for a more accurate analysis of spots on the surface of a thin-layer chromatography plate. By superimposing several digital images with the correlated positional information of their respective pixel coordinates, and referencing them to the same real-world coordinates of the thin-layer chromatography plate, it is possible to identify spot regions on the thin-layer chromatography plate and their corresponding images that are invisible to the human eye but can be considered in the evaluation of the thin-layer chromatography plate.

[0010] According to aspects of the present invention, at least one digital image is captured using illumination of a thin-layer chromatography plate with visible light, and at least one digital image is captured using illumination of a thin-layer chromatography plate with ultraviolet light. Visible light may be monochromatic light having wavelengths in the wavelength range of about 400 nm to 750 nm, or between 380 nm and 800 nm. Visible light may also be light from an illumination device with broad emission characteristics, for example, white light including wavelengths between 400 nm and 800 nm. Ultraviolet light includes wavelengths less than 400 nm, preferably less than 300 nm. Ultraviolet light may be emitted from a gas discharge lamp. However, in relation to size and energy consumption, illumination devices used for different illuminations may include light-emitting diodes with suitable emission characteristics.

[0011] For many samples, it is advantageous to use two digital images with significantly different illuminations: one digital image taken using visible light illumination and another digital image taken using ultraviolet light illumination. As a result, many spots that are not very noticeable or visible in one digital image become clearly visible and easier to identify in the other digital image. Furthermore, for many solvents combined with many thin-layer chromatography plates, the position of the mobile phase on the solvent front after the thin-layer chromatography plate has been developed is better visible with ultraviolet light, for example, with a wavelength of 254 nm, compared with visible light illumination. By superimposing the digital image information from both digital images, information from the less significant digital image is also considered, allowing for further evaluation and analysis of the sample information.

[0012] In yet another preferred embodiment of the present invention, at least three different digital images are captured using different light illuminations and used for evaluation. The three different illuminations may include, for example, illumination using white light, illumination using ultraviolet light with a wavelength of 366 nm, and illumination using ultraviolet light with a wavelength of 255 nm. For many samples, it has been found that illumination with two different wavelengths within the ultraviolet region results in significantly different digital image information. Thus, by superimposing digital images of two different ultraviolet illuminations combined with visible light illumination, the amount of information in the superimposed image is significantly improved. For many applications, ultraviolet illumination with a wavelength of 270 nm or 310 nm is also suitable and provides additional information that can be used for evaluating thin-layer chromatography plates. It is also possible to superimpose two different illuminations within the visible light region, for example, digital images using either blue light or red light illumination. Furthermore, different illuminations may also be adapted to the specific characteristics of the camera or optical imaging system used to capture different digital images of the thin-layer chromatography plate. Thus, by using a color CCD with high sensitivity in the red, green, and blue regions, for example, two or more different illuminations can be used to match the high sensitivity of the optical imaging system, resulting in high image intensity in the corresponding red, green, or blue wavelength ranges of the color CCD.

[0013] During the evaluation step, image information from two or more digital images is superimposed. The image information may include any information that can be extracted by analyzing the digital images, such as the location and intensity of spots of a sample separated on the surface of a thin-layer chromatography plate. However, according to an advantageous aspect of the present invention, the image information used for superimposition is equal to or proportional to the pixel values ​​of the pixels in the digital images. The pixel values ​​may be intensity values ​​or color-specific values ​​of the pixels in question from the digital images. Thus, superimposition can be performed without any prior analysis of the digital images and without any manual support. According to another aspect of this feature, pixel values ​​may be calculated based on such pixel information from the digital images, allowing for the addition of additional information, such as the type of lighting used to capture the digital images, or different weighting information between different areas on the surface of the thin-layer chromatography plate. It is also possible to perform some image analysis and automated image enhancement on each individual digital image before superimposing the pixel values ​​of each digital image. Instead of pixel values, it is also possible to superimpose image information based on information from areas of the digital images, such as the average intensity of the spot areas in the digital images. The resulting overlay includes the image information of all the digital images considered for superimposition. Evaluating and analyzing superimposed overlays is performed with greater accuracy and typically yields better results than evaluating and analyzing each individual digital image. Extracting image information used to superimpose two or more digital images, as well as the subsequent superimposition of corresponding image information, can be done automatically, for example, based on predefined methods and thresholds.

[0014] Each pixel value can be in any color-specific format or file format used to store the digital image and the resulting superimposed image information. For most applications, the superimposed image information may be proportional to the sum of all different colors, i.e., the sum of the image information across all wavelengths. However, it may sometimes be advantageous to restrict the image information to a predetermined color gamut or to a single color. For example, the image information may be selected as the green component of the pixel color in a digital image. The superposition may be performed over the entire surface of the digital image, preferably over the entire surface of the thin-layer chromatography plate identified in the digital image. It is also possible to limit the superposition to only the area of ​​the thin-layer chromatography plate surface that contains a spot identified in at least one digital image.

[0015] In yet another embodiment of the present invention, the image information is proportional to the grayscale values ​​of the pixels within each region. It is considered preferable to capture grayscale images from thin-layer chromatography plates or to convert digital images captured in different color formats into grayscale format. Typically, there is no significant loss of image information, but the computational load required for overlay and subsequent evaluation can be reduced.

[0016] According to an advantageous aspect of the present invention, in the evaluation step, a track identification step is performed, thereby determining the stripe intensity as the sum of the pixel intensities of the pixels within each stripe for a predetermined number of stripes flowing from the baseline to the solvent front, and thereby identifying the corresponding track for each stripe having a local maximum or local minimum of stripe intensity. The number of tracks can be manually preset by a user who identifies the correct number of tracks laid out on the thin-layer chromatography plate. It is also possible to provide an automated determination of the number of tracks using automated image analysis based on pixel intensity, for example, which is more convenient for the user. The general shape and orientation of the tracks can be preset to facilitate image analysis performed by appropriate software. However, in most applications, since most spots are elliptical with their longest length direction perpendicular to the direction of the track (usually a track contains several such spots), it is possible to automatically identify tracks based on the shape and orientation of the spots located within a single track. Depending on the type of thin-layer chromatography plate and the lighting used to create the digital image, the spots will be visible as bright areas on a dark background or dark areas on a bright background. Therefore, the stripe intensity can be either locally maximum or locally minimum when compared to the intensity of the region adjacent to each stripe.

[0017] According to another preferred aspect of the present invention, the evaluation of thin-layer chromatography involves the calculation of concentration measurement information along a predetermined line or track along a thin-layer chromatography plate. While a thin-layer chromatography plate is being developed with one or more samples, each sample will be separated along a predetermined linear track on the surface of the thin-layer chromatography plate. A track is a transversely elongated area on the surface of the thin-layer chromatography plate that is covered by the separation of the sample material during development. The direction of the track is equal to the direction of movement of the mobile phase during the development of the thin-layer chromatography plate. The width of the track is equal to the width of at least one spot visible along the track, which usually corresponds to the width of the sample material coated on the thin-layer chromatography plate along this track. If several spots are visible along the direction of separation of the sample material, the width of the track may be fitted to either the maximum width of any of the spots along this track, or an average width value calculated by calculating the average value of all the respective widths of all the spots along this track. Thus, this information can be used to focus on image information along such a track. It is possible to identify each track for each sample by summing all the pixel values ​​along the direction of the track, i.e., along the direction of movement of the mobile phase during the development of the thin-layer chromatography plate and the separation of the sample material. The expected minimum track width can be preset as the number of pixels corresponding to that minimum track width. The total pixel value, which is the result of a sum operation along the track direction, is then calculated along a direction perpendicular to the track direction. If the total pixel value exceeds the preset minimum track level or is less than the preset maximum track level, at least for the minimum track width, this stripe along the track direction is identified as a sample track, and the sample is separated along this sample track within it. Evaluation and analysis of the digital image, or a superposition of two or more digital images, can then be focused on or limited to all identified sample tracks.

[0018] According to aspects of the present invention, the position of a thin-layer chromatography plate in a digital image is identified by automated digital image processing. Such identification can be performed using a well-known software module or method that can be easily implemented within a software program, which performs some or all of the steps necessary for evaluating a thin-layer chromatography plate, namely the digitization step, the position identification step, and the evaluation step, including the superposition of two or more digital images and automated track detection. In addition to identifying the position of the thin-layer chromatography plate, the size and orientation of such plate can also be determined by automated digital image processing.

[0019] The identification of thin-layer chromatography plates in digital images, or the number of tracks, track orientation, and track width, is performed by the automated digital image processing steps described above, and recommendations for each identification result are made as a result, which can be visualized for review, confirmation, or correction by the user. Thus, if the automated identification of a thin-layer chromatography plate, or the identification of one or more tracks on it, fails, the user can manually support or confirm each identification. However, in most cases, the user will be able to review the results of the automated identification of thin-layer chromatography plates or tracks in the digital image. Furthermore, according to yet another aspect of the present invention, it is possible to remove artifacts from the digital image within the image cleaning step, either before or during the evaluation step. The size and shape of the spots on the sample material can be approximated. Areas that are darker or brighter than the clean surface of the thin-layer chromatography plate can be compared to preset parameters that define the spots, and all such areas that do not match the spot criteria can be discarded as artifacts. Since all spots must be located within the track, it is also possible to utilize already determined track information and discard all darker or brighter areas outside the track.

[0020] It is also possible to remove artifacts within a spot. By utilizing known image noise reduction algorithms, artifacts within a spot can be identified, and appropriate correction can be performed, for example, by replacing the pixel intensity affected by the artifact with the pixel intensity of the adjacent portion of the spot that is not affected by the artifact.

[0021] Many of the steps described above can be performed using electronic data processing devices. For most steps, suitable and appropriate software programs can be executed on standard data processing devices. For example, it is also possible to use individually tailored hardware configured to perform automated image analysis on large digital images in a short amount of time.

[0022] The present invention also relates to an evaluation device for thin-layer chromatography, comprising a light-resistant, openable digitization chamber with a base plate for receiving a thin-layer chromatography plate, an illumination device for illuminating the thin-layer chromatography plate on the base plate, a digital imaging device for obtaining a digital image of the thin-layer chromatography plate illuminated by the illumination device, and an evaluation unit for evaluating the digital image. Evaluation devices described in the prior art or available for evaluating thin-layer chromatography plates using separated samples are typically limited to a few basic evaluation steps that can be performed on a thin-layer chromatography plate of a given size. In most cases, evaluation and further analysis of thin-layer chromatography plates require manual input and interactive processing with a user who has significant skill and experience in evaluating thin-layer chromatography plates. Therefore, there is a need for evaluation devices that provide additional support for the evaluation of thin-layer chromatography plates and enable some or all of the necessary steps in the evaluation process to be performed automatically.

[0023] According to an advantageous aspect of the present invention, the evaluation device is adapted to perform the method described above. The evaluation device includes two or more different illumination devices adapted to illuminate a thin-layer chromatography plate inserted into a digitization chamber using light of different wavelengths. Furthermore, the evaluation device includes an evaluation unit adapted and preferred to perform the evaluation method described above, namely, capturing two or more different digital images from a thin-layer chromatography plate inside the digitization chamber using different illumination, and correctly superimposing the image information of the two or more different digital images. The evaluation unit may also preferably perform a digital image processing method to automatically identify the size, position, and orientation of the thin-layer chromatography plate. According to another aspect of the present invention, the evaluation unit is also designed and configured to identify tracks within the surface of the thin-layer chromatography plate and remove artifacts that do not conform to preset criteria defining spots of separated samples. Using such an evaluation device, a fully automated evaluation of different thin-layer chromatography plates can be performed without requiring manual input or user interaction. Of course, it is also possible to display or save the results of each single step performed during the evaluation to allow for subsequent review of the evaluation process or manual input or interaction if deemed preferable during or after the evaluation process.

[0024] According to another aspect of the present invention, the evaluation device includes a data communication unit that enables wireless data transmission of data relating to the evaluation process to a thin-layer chromatography data storage device or other devices that may be used during the evaluation of a thin-layer chromatography plate. Thus, all information and data that may be relevant to the evaluation of a thin-layer chromatography plate can be transmitted to a storage device for storing this information and data for future use, for example, to review or repeat the evaluation process. Furthermore, the stored information and data can be easily accessed and retrieved from anywhere in the world. Thus, the results of the evaluation of a thin-layer chromatography plate can be analyzed and compared with other data at any location and time, and are not limited to the location of the evaluation device. In addition, since the evaluation results can be easily accessed and converted to different data formats without the need to physically access the thin-layer chromatography plate on which the digital image was taken, it is also possible to perform different types of data analysis using different devices or at different locations and times. Furthermore, the digital image and corresponding pixel information, or information about spots on the thin-layer chromatography plate, can be transferred together with the thin-layer chromatography plate to another device that may be used for further analysis of the thin-layer chromatography plate. For example, a thin-layer chromatography plate can be transferred to an automated mass spectrometer that can automatically remove some sample material from preset spot areas within the thin-layer chromatography plate. The removed spot areas, for subsequent analysis of the sample material, are included in the evaluation results of the thin-layer chromatography plate and may become information identified, selected, and preset by an automated method pre-performed using the evaluation device based on these evaluation results.

[0025] Data transfer can be triggered manually or performed automatically. The data communication unit may include a standardized communication interface such as, for example, a WLAN interface, a Bluetooth interface, or an NFC interface. It is also possible to use an individually configured data communication unit that enables data transfer from an evaluation device to a storage device near the evaluation device, i.e., in the same location, or far away from the evaluation device, i.e., in a different location or country. Data transmission can be provided by using a first data transmission protocol for the first data transmission from the evaluation device to an additional data transmission device that functions as a router and is located near the evaluation device, i.e., in the same room, and then using a second and different data transmission protocol to transmit the data from the additional data transmission device to a storage device that can be located anywhere and far away from the evaluation device.

Brief Description of the Drawings

[0026] The present invention will be more fully understood and further features will become apparent when reference is made to the following detailed description and the accompanying drawings. The drawings are merely representative and are not intended to limit the scope of the claims. In fact, those skilled in the art may recognize that various modifications and variations can be made without departing from the innovative concept of the present invention when reading the following specification and viewing the drawings. Similar parts depicted in the drawings are referenced by the same reference numerals.

[0027] [Figure 1-a] FIG. 1 illustrates a schematic view of two digital images of the same thin layer chromatography plate, each illuminated with a different light, thereby showing the spots of the sample material visible in each illumination. [Figure 1-b] FIG. 1 illustrates a schematic view of two digital images of the same thin layer chromatography plate, each illuminated with a different light, thereby showing the spots of the sample material visible in each illumination. [Figure 2]Figure 2 shows a schematic view of two digital images superimposed after digital image processing to identify the size, position, and orientation of a thin-layer chromatography plate. [Figure 3] Figure 3 shows additional representations of the separated sample material tracks, as well as a magnified schematic view of the superimposed image shown in Figure 2. [Figure 4] Figure 4 illustrates the sum of pixel values ​​along a line parallel to the baseline of the sample material and perpendicular to the direction of solvent movement, while showing track identification during the development of the thin-layer chromatography plate. [Figure 5] Figure 5 illustrates the concentration measurement information along the single track shown in Figure 3. [Figure 6] Figure 6 shows a schematic cross-sectional view of an evaluation device according to the present invention, which has been designed and configured to perform the evaluation method according to the present invention.

[0028] Figure 1, on the left, shows the first digital image 1 of a thin-layer chromatography plate 2 with three different samples separated along the direction of movement indicated by arrow 3. The first digital image 1 was taken inside a light-resistant digitization chamber within the evaluation device 4, which is illustrated in more detail in Figure 6. During the acquisition of the first digital image 1, the thin-layer chromatography plate 2 was illuminated with visible light, i.e., white light with a broad emission range in the wavelength range between 400 nm and 800 nm. Figure 1, on the right, shows the second digital image 5 of the same thin-layer chromatography plate 2 illuminated with ultraviolet light, i.e., light with a narrow wavelength band around 366 nm. Thus, after the digitization step, two digital images 1 and 5 are available, containing images of the same thin-layer chromatography plate 2 taken using different illumination. During the unfolding of the thin-layer chromatography plate 2, which took place before the two digital images 1 and 5 were taken, each of the three samples was exposed to the solvent, moving from an immersion area adjacent to the baseline, which is normally parallel to the lower edge 6 of the thin-layer chromatography plate 2, along the movement direction 3 across the surface 7 of the thin-layer chromatography plate 2, toward the solvent front near the upper edge 8 of the thin-layer chromatography plate 2. The components of the three samples are separated into distinct spots 9 and 10 along the movement direction 3. However, due to different lighting, some spots 9 and 10 are visible in only one of the two digital images 1 and 5, while some other spots 9 and 10 are visible in both digital images 1 and 5. In addition to the spots 9 and 10, within each of the digital images 1 and 5, there are also artifacts 11 and 12 that do not imply separated sample material, but are generated by, for example, dust or other contaminants on the thin-layer chromatography plate 2. In the position identification step, the size and position of the thin-layer chromatography plate 2 in digital images 1 and 5 are determined by digital image processing using, for example, a well-known method for determining the boundaries of a transverse shape of any size and orientation in digital images 1 and 5. Then, for each of digital images 1 and 5, the image of the thin-layer chromatography plate 2 is referenced to coordinates on the surface 7 of the thin-layer chromatography plate 2.

[0029] During the subsequent evaluation steps, two images of thin-layer chromatography plate 2 from two digital images 1 and 5 are first overlaid by superimposing their corresponding pixel values. The overlay of pixel values ​​for the superposition of the two digital images 1 and 5 can be performed, for example, by adding the pixel values ​​from the two digital images 1 and 5, or by adopting the maximum pixel value, or by calculating a weighted sum of the pixel values, so that each weighting coefficient can be preset or determined based on any available digital image information. The resulting overlay, or superposition 13, is illustrated in Figure 2. Superposition 13 shows all the spots 9 and 10 visible during either visible light illumination or ultraviolet illumination. Since the size and position of the images of thin-layer chromatography plate 2 in the two digital images 1 and 5 are pre-matched, the positions of the spots 9 and 10 also match. Thus, superposition 13 contains more information about the sample, as each of the respective digital images 1 and 5 contains.

[0030] Figure 3 illustrates the superposition 13 of two images of the thin-layer chromatography plate 2 in more detail and in a magnified view. During the track identification step, for a number of stripes flowing along the movement direction 3 from the baseline, i.e., the lower edge 6, to the solvent front, i.e., the upper edge 8, the normalized stripe intensity I / I0 is determined as the sum of the pixel intensities of the pixels within each stripe, thereby identifying the corresponding track for each stripe with a local maximum of stripe intensity I / I0. The movement direction 3 of the thin-layer chromatography plate 2 is aligned along the Y-axis of the coordinate system referenced by the thin-layer chromatography plate 2. Each stripe flows parallel to the Y-axis. For each position or pixel along the line flowing parallel to the X-axis, the summed stripe intensity I / I0 is shown in Figure 4. The stripe intensity I / I0 can be calculated by converting the superposition 13 to a grayscale image and by adding up the grayscale values ​​of each pixel along each stripe. To identify tracks a, b, or c, several appropriate criteria can be preset, such as the minimum width or number of stripes forming the track, and the minimum stripe intensity threshold or minimum track level 14 of the required stripe intensity I / I0 for the track. Based on the calculated stripe intensity I / I0, tracks a, b, and c can be identified as ranges of pixels with a stripe intensity I / I0 that satisfies all the criteria. Tracks a, b, and c identified in this manner are shown as dashed lines in Figures 3 and 4.

[0031] After the tracks a, b, and c corresponding to each sample separated along the corresponding tracks a, b, and c are identified, track concentration measurement information can be calculated by summing all pixel values ​​in tracks a, b, or c with the same Y-axis value, i.e., across the corresponding tracks a, b, or c, along the movement direction 3, i.e., along the Y-axis. Figure 5 illustrates such track concentration measurement information calculated for track b in Figure 3. Such track concentration measurement can serve as a basis for further analysis of the results of sample separation performed using the thin-layer chromatography plate 2. During the image cleaning step performed between the evaluation steps, artifacts 11 and 12 are compared against predefined criteria, as well as track information relating to previously identified tracks a, b, and c. Artifact 11 is outside of tracks a, b, and c and is therefore discarded. Artifact 12 is inside track c but does not meet the predefined criteria regarding the shape and size of spots 9 and 10 and is therefore discarded.

[0032] In Figures 3, 4, and 5, the different hatchings of spots 9 and 10, stripe intensity I / I0, and track density measurements show their respective contributions from two different digital images 1 and 5 to the resulting superposition 13, and the information calculated based on the superposition 13.

[0033] Figure 6 illustrates an exemplary embodiment of the evaluation device 4. The evaluation device 4 includes a light-resistant, shielded housing 15 surrounding the digitization chamber 16, which is mounted inside the digitization chamber 16 in a slidable manner and includes a base plate 17 that can move linearly out of the digitization chamber 16 to receive and remove the thin-layer chromatography plate 2.

[0034] The evaluation device 14 further includes a first illumination device 18 for illuminating the thin-layer chromatography plate 2 using visible light emitted from a light-emitting diode, and a second illumination device 19 for illuminating the thin-layer chromatography plate 2 using ultraviolet light with a wavelength of 366 nm emitted from a corresponding light-emitting diode. The first illumination device 18 and the second illumination device 19 may also be gas discharge lamps or other illumination devices that emit light and illuminate the thin-layer chromatography plate 2 placed on the base plate 17. The evaluation device 14 also includes a digital imaging device 20 for taking digital images 1, 5 from the thin-layer chromatography plate 2 illuminated using either the first illumination device 18 or the second illumination device 19.

[0035] Digital images 1 and 5 are processed using an evaluation unit 21 that performs the evaluation method described above. All image information and additional data are sent to a data communication unit 22 and may be transmitted to a data storage device not shown in Figure 6. The evaluation device 14 also includes a display 23 for displaying relevant information to the user. The display 23 may be a touch sensor display that allows for some user input, for example, some trigger information to perform some or all steps during the evaluation process.

Claims

1. A method for evaluating a thin-layer chromatography plate (2) after a separation process for separating the components of a sample on the thin-layer chromatography plate (2), wherein in a digitization step, digital images (1, 5) of the thin-layer chromatography plate (2) are captured using a digital imaging device (20); thereby, in a position identification step, the position of the thin-layer chromatography plate (2) in the digital images (1, 5) is identified; thereby, in an evaluation step, the positions of visible spots (9, 10) indicating the sample components on the thin-layer chromatography plate (2) are determined; and the thin-layer chromatography is evaluated based on the determined positions of the spots (9, 10). The digitization step involves capturing at least two digital images (1, 5) that differ in terms of the wavelength range of illumination; the position identification step involves identifying the position of the thin-layer chromatography plate (2) for each of the at least two digital images (1, 5); and the evaluation step involves superimposing the image information of the at least two digital images (1, 5) over at least all regions having at least one visible spot (9, 10) in at least one of the at least two digital images (1, 5), and the superimposition (10) of image information from the same region of the at least two digital images (1, 5) can be used to evaluate the thin-layer chromatography plate. The present invention is characterized in that at least one digital image (1) is captured using illumination of a thin-layer chromatography plate (2) using visible light, and at least one digital image (5) is captured using illumination of a thin-layer chromatography plate (2) using ultraviolet light. The aforementioned method.

2. The method according to claim 1, characterized in that at least three different digital images (1, 5) are captured using illumination with different types of light and used for evaluation.

3. The method according to claim 1 or 2, characterized in that the image information used for superimposition is equal to or proportional to the pixel values ​​of the pixels in the digital image.

4. The method according to claim 3, characterized in that the image information is proportional to the grayscale value of the pixels within each region.

5. The method according to one of claims 1 to 4, characterized in that the evaluation of thin-layer chromatography includes the calculation of concentration measurement information along predetermined lines or tracks (a, b, c) along a thin-layer chromatography plate (2).

6. The method according to one of claims 1 to 5, characterized in that the position of the thin-layer chromatography plate (2) in the digital image (1, 5) is identified by automated digital image processing.

7. The method according to one of claims 1 to 6, characterized in that artifacts are removed from the digital images (1, 5) during the image cleaning step, either before or during the evaluation step.

8. The method according to any one of claims 1 to 7, characterized in that, within the evaluation step, a track identification step is performed, thereby determining the stripe intensity as the sum of the pixel intensities of the pixels within each stripe for a predetermined or automatically detected number of stripes flowing from the baseline to the solvent front, and thereby identifying the corresponding track (a, b, c) for each stripe having a local maximum or local minimum of stripe intensity.

9. A thin-layer chromatography evaluation device (14) comprising a light-resistant open / close digitization chamber (16) having a base plate (17) for receiving a thin-layer chromatography plate (2), illumination devices (18, 19) for illuminating the thin-layer chromatography plate (2) on the base plate (17), and a digital imaging device (20) for obtaining digital images (1, 5) of the thin-layer chromatography plate (2) illuminated by the illumination devices (18, 19), and an evaluation unit (21) for evaluating the digital images (1, 5), One type of lighting device uses visible light, and the other uses ultraviolet light. The evaluation unit is for evaluating the superposition of image information from the same region of at least two digital images (1, 5) that differ in terms of the wavelength range of illumination. The aforementioned device.

10. The evaluation device (14) according to claim 9, characterized in that it includes a data communication unit (22) that enables wireless data transmission of data relating to the evaluation process to a thin-layer chromatography data storage device.

Images