JPS59120939A - Quantitative evaluating device for thin-layer chromatogram - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention The present invention is for quantitatively and automatically evaluating a thin layer chromatogram by recording light reflected or emitted by a developed thin layer plate using a densitometer K. The apparatus comprises: a light source, means for forming a line image of light on the laminar plate, means for effecting a relative movement between the line of light and the laminar plate, reflection by the laminar plate; or comprises an optical unit for recording the emitted light and means for converting, storing and evaluating the analog signal obtained when recording its reflection or emitted light with a parameter proportional to the amount of matter. .

Quantitative evaluation of thin layer chromatograms is currently performed essentially by three methods. The most commonly used method is to direct a line of light against a TLC plate such that a series of TLC spots are recorded as centrally as possible, i.e. the focus of their density profile is in the center of the projected line. Consists of moving. The signal obtained when measuring the light reflected by the linear measurement area is correlated with the substance concentration.

This method has a number of drawbacks, not least of which is the presence of material spots of extremely variable shape and size within the chromatographic path to be measured, and because of these the length of the line, which is the largest material (must be selected according to the size of the spot) and the fact that the width of the line cannot be adjusted to an optimal value for all material spots. Since the concentration profile of a TLC spot generally along the length of the line is necessarily non-linear, the densitometer signal does not depend on the substance concentration in the TLC spot to an extent that can be mathematically defined and therefore provides a quantitative evaluation. It is necessary to collect a calibration curve that must be linearized by calculation. This is only possible within a relatively narrow concentration range in each case. The necessary optimization of the line size and line position for each material spot to be measured is itself extremely time consuming.

Some of these drawbacks of devices using line scanning can be avoided with spot scanning devices. In this case, the surface of the TLC plate is scanned with light spots in a meander or zigzag pattern. Since the area of this light spot must be extremely small compared to the material spot, a complete scan of the TLC plate requires a considerable amount of time. The point of light must be guided very precisely, which poses considerable mechanical and technical control problems. Since baseline determination can only be performed at each point where the meander or zigzag movement changes direction, changes in the baseline along the path of the point of light are not recorded, which can lead to considerable errors in the quantitative evaluation. be.

Recently, a device has been developed that simultaneously images the entire TLC plate on a screen using a video camera. However, in this case as well, strict quantitative evaluation is difficult. That is, it is extremely difficult to uniformly irradiate the entire area of one TLC plate, and it is actually impossible with monochromatic light. If the local resolution in both dimensions is high, the true concentration within the TLC spot is true.''1] Extremely fast with short time resolution of the constant distribution and extremely large working memory requiring a resolution of more than 500 kilobytes for 8 humans. Analog-to-digital converters would be required, but they would involve considerable expense.

Therefore, it is an object of the present invention to find a quantitative evaluation device for thin-layer chromatograms that allows evaluation to be performed extremely quickly, with high precision, and as automatically as possible.

Now, for this purpose [the TLC spot is scanned with a line of light in a manner known per se, a large number of locally arranged optical We have found that this can be achieved by recording the light reflected from the sensor K (so the first integration can be performed by each individual sensor, where extreme local resolution is required). The output signal obtained by each sensor can then be converted using a computer in a manner known per se into a ξ parameter proportional to the amount of substance, and this /ξ parameter can then be stored in a data storage device. , and after all TLC, Zbots have been scanned, their S parameters can be summed to the final value.

That is, the present invention provides a light source, a means for forming a line image of light on the laminar plate, a means for effecting relative movement between the line of light and the laminar plate, a light source reflected or emitted by the laminar plate, an optical unit for recording the light emitted by
reflected or emitted by the unfolded laminar plate and includes means for converting, storing and evaluating the analog signal obtained in recording the light reflected or emitted by it with a parameter proportional to the amount of material. This is a device for quantitatively and automatically evaluating thin-layer chromatograms by recording light, and the optical unit for recording the light reflected or emitted by the thin-layer plate includes a number of optical centers. The IR area of the thin layer route 10 recorded by each sensor is small enough that the density printing distribution within this area can be considered uniform; The signals obtained can be converted into parameters proportional to the amount of material corresponding to the individual area elements, these parameters can be stored in a data storage means, and all segments of the material spot on the thin plate can be recorded. After that, a transducer, storage means, computer and readout unit are provided which can aggregate all the transformed and stored ξ parameters to give a single value corresponding to the amount of material in the observed thin layer spot. The present invention relates to an automatic quantitative evaluation device for thin layer chromatograms.

A significant advantage of the inventive device is that it makes it possible to simultaneously record the baseline on both sides of the spot, so that for each measuring position, before converting into a parameter proportional to the amount of material that should be distributed to that baseline, All m l#: #+ times the signal can be corrected by the signal. Another substantial advantage is that the concentration profile along the line of light is resolved into a large number of individual measuring points1, resulting in homogeneity within the area segment observed by each individual cell. The signal obtained by the sensor also has a mathematically definable relationship to the concentration of the material coating at each location.

The drawings schematically show a part of a preferred embodiment of the device of the invention.

In these figures, 1 indicates the TLC plate and 2 indicates the material spot developed on it. 3i-
i' is a monochromator (monochromatic light source), 4 is a line of light, and 5 is an individual light transmission (propagation) body provided with a guide track 6. 7 indicates a photodiode, 8 indicates a focusing optical means and 9 a line array;
For example, a self-scanning linear photodiode 1c is shown. As parts of the optical transmission system, 10 indicates a central fiber and 11 indicates a casing fiber, both of which are housed together in a guide track 12. 13 is a multi-element light receiver, 14 is a polychromator (polychromatic light source), and 15 is a plane in which the light receiver 13 can move. 16 is a lamp, 17 is a slit, 18 is a converging lens, 19 is a holographic grating, and 20 is a two-dimensional multi-absorption receiver.

There are many possibilities for implementing the features of the invention. In each case, the developed TLC plate (1) with the material spots (2) is illuminated with a linear band of light (4);
and adjusting the relative movement between the light line (4) and the TLC plate (1) so that at least a partial area of the surface of the TLC plate (1) is scanned without gaps during the course of the evaluation. do. This relative motion can be in the flow direction of the chromatogram or across it;
Thereby, chromatographic development spots (material spots in the direction of development of the sample) or material spots of different samples with the same Rf value are evaluated in each step. The means for effecting this relative movement are known to those skilled in the art and need not be explained here.

A device using monochromatic light is shown in FIGS. 1-4.

The light sources used can be, for example, low- or high-pressure mercury lamps, deuterium lamps, xenon lamps or tungsten lamps.

The light source, monochromator and TLC plate (1)
Means for producing a monochromatic light image as a line of light (4) on top are known to those skilled in the art and also need no further explanation.

An optical sensor for segmentally recording the light reflected or emitted from the TI, C plate (1) is used to record the light a (
It is preferable to arrange them in a line parallel to the image 4). In order to effectively decompose the observed area into individual segments, this line should consist of at least five individual segments.

As shown in FIG. 1, if a photodiode (7), such as a photovoltaic recon diode or a recon diffusion bin photodiode, is used as the sensor, the line will be 5 to 50. It should preferably consist of about 20 diodes. Other optical sensors, such as self-scanning linear arrays (9) with up to 1,000 or more elements per line, such as photodiodes or charge-coupled devices or charge-coupled image sensors (CC
The D string is also known. 50-1. []0[J especially about 2
Preferably, a linear array with 50 side force elements is used.

The number of processors used also matches the length of the light line (4). The area element observed by one sensor is approximately υ015
It should have an edge length of ~0.8m. Therefore, when using 5 setsa, the length of the light line (4) is 3
~Amm should not be exceeded.

Therefore, when measuring a wider chromatographic spot, it is preferable to use a larger number of sensors. The number of sesors is preferably selected such that the edge length of the area element observed by one sesser is approximately [ ], 005 to 0.2 m.

The transmission of the light reflected or emitted by the TLC play 1- (11 can be accomplished, for example, using photoconductors (5, 11) as shown in FIGS. 1 and 6, or as shown in FIG. This can be done using a focusing optical means (8).As shown in FIG. A central fiber consisting of a fiber bundle' (
1o) Used to illuminate with light emitted from the Id monochromator (3), while the casing fiber (11)
The reflected or emitted light is transferred to a multi-element receiver (13) K
Helpful for transmission. Preferably, the optical conductors (5, 11) are each connected to an individual photodiode.

A quartz optical transmission fiber can be used as the optical transmission body, and is preferred. focusing optical means (8);
For example, a converging lens or a concave mirror is preferably used in combination with the linear array (9).

As shown in Figures 1 to 3, TLC Brake)
(1) can be irradiated with monochromatic light. This is made using a conventional monochromator, such as an interference filter or grid monochromator. If the illumination is performed with polychromatic light, a wider range of information can be obtained. When using a polychromator, such as a grid monochromator (minus the exit slit) or a holographic-grating polychromator, each light wavelength is aligned perpendicular to the imaged line of light and thus to the axis of the optical sensor. It is also converted to position coordinates perpendicular to the object. Such a device is shown in FIG. 4, in which the measured wavelength can be adjusted by varying the position of the multi-element receiver (10) in the focal plane (15) of the polychromator (14). I can do it.

Such a device has the advantage that there is no need to use a monochromator as a light dispersion element, so that the light source output has to meet even fewer requirements. For example, instead of the usual twin light source using a mercury lamp or deuterium lamp for the V range and a tungsten lamp for the visible range, a single light source of lower output power, such as a 50 to 100 watt Gysenon lamp, can be used. You can also do it. Furthermore, the polychromator can be arranged such that the light reflected or emitted by the TLC blade 1)K is recorded at a much wider solid angle than was possible in the prior art. This allows changes in reflectance or fluorescence to be detected with higher sensitivity. Furthermore, the construction on the concentration side is extremely simple. A high performance densitometer can be made using a minimum of optical components.

A linear multi-element light receiver disposed movably in the direction of the light receiving surface (15) (instead of 16 linear multi-element light receivers, several linear multi-element light receivers are arranged in parallel within the light receiving surface, and each light receiving device In such a case, a two-dimensional multi-element light receiving device (20) as shown in the device of FIG. 6 can be used, and it is preferable. 18) through the slit (17) using the lamp (
The light beam (4) projected from 16) onto the TLC plate (1) is resolved over the entire wavelength range by a holographic grating (19).

In this way, density-side measurements can be made simultaneously for several wavelengths or all wavelength ranges, and there is no need to change the light source to record different wavelength ranges. Not only is the evaluation more rapid, but this also allows substances that have been incompletely separated by chromatography to be further chromatically separated insofar as the absorption profiles of the substances differ. In the preferred use of a two-dimensional multi-element receiver (29), e.g. a photodiode matrix or a video camera, any mechanically actuated optical component that requires adjustment and maintenance. is no longer necessary at all. Instead of recording the entire scale range, the desired scale range can be selected without any problem by correspondingly programming the connected computer.On the other hand, the complete With the ability to obtain on-screen images in milliseconds, the necessary information critical to optimizing the field evaluation is available very quickly.

These advantages are not limited to reflectance measurements, but can also be applied to fluorescence measurements. Certain absorbing wavelengths can be removed from the light source by increasing the fill, and in both cases correspond to the fluorescence range. ] The first wavelength automaton is filtered out again before entering the polychromator, or suppressed during the evaluation, so that the logic region is evaluated.

Significant advantages in terms of time are obtained when using dual-wavelength deconcentration, which is known per se and is extremely time-consuming in optimizing the measurement wavelength and the standard wavelength. This is because, for example, all the information appears on a screen connected to the device within a few seconds, and the wavelength of absorption maximum can be determined immediately as the measurement wavelength and a suitable wavelength outside the absorption band as the standard wavelength. be. The device of the invention ensures that both information items always originate from the same point on the TLC plate and are obtained at the same time.

In prior art devices, this required extremely complex optical equipment and could only record individual bulletin items alternately.

For example, with the device according to the invention shown in FIG. 6, it is also possible for the first time to carry out operations using at least two standard wavelengths, or even more standard wavelengths, in addition to the measurement wavelength.

Since the absorption and scattering of the coating material on the TLC plate is wavelength dependent, background noise cannot be completely eliminated if only one standard wavelength is used. However, if at least two standard wavelengths (preferably chosen at short and long wavelengths relative to the measurement wavelength) are used, the absorption and scattering linearity of the coating material over this wavelength range is determined by the Kutle process. Noise can be virtually completely eliminated when recording.

The present invention is aimed at recording measurement values necessary for densitometer evaluation. The conversion of the obtained analog signals into digital measured values and their storage, addition and evaluation can be carried out by means and methods known per se. This requires a linear array or a seven-trick array (two-dimensional array) or an analog-to-digital converter with a computer-acceptable output, computer and software.

The device according to the invention makes it possible for the first time to obtain measurements that are independent of the position, size and shape of the material spot (2). This eliminates the need to optimize the position of the individual substance spots (2) in the light line (4) manually or by means of expensive computerized control devices, and allows for a linear dependence of the measurement signal on the substance concentration. It is possible to achieve
2) is independent from the shape of TL, which has a concentration band.
Both the C plate and the plate developed two-dimensionally several times can be evaluated without any problem. With the device of the invention, a clear distinction can be made between spot areas and substance-free background areas, which are excluded by the computer from all measurements. Therefore, in contrast to known evaluation devices, the width of the line is no longer critical (I), and the difference in the Sidanol/noise ratio due to spot size is completely eliminated.

By decomposing the reflectance or luminescence values into individual measurement points along the line of light (4), it is no longer necessary to completely separate from each other the substances present along the line. This therefore provides the possibility of interrogating the TLC plate (1) with light a (4) in the direction of the chromatography. This is because unresolved material peaks can be solved by a computer (3) by methods similar to those known for gas chromatography and liquid chromatography. In the same way, it is possible to position the deployment spots of the U matogranoi so close to each other that they are adjacent but not yet touching. It is even possible to choose the length of the light line (4) so that several chromatographic development spots can be recorded simultaneously. Known evaluation devices do not allow either of these methods.

As described above, the evaluation device of the present invention can disturb the deployed TLC brake 1 and quickly evaluate it with precision that has not been achieved until now.
provide an advantageous evaluation method.

[Brief explanation of drawings]

Figure 1 shows the image of a line of light created by monochromatic light on a TLC plate.
A device is shown in which the light reflected or emitted from the surface is transmitted via a light conduit to a receiver. FIG. 2 shows a similar device in which the light reflected or emitted from the surface is directed into a linear array by focusing optical means. FIG. 3 shows a device in which both the illumination of the TLC plate and the guidance of the reflected or emitted light is carried out using a light conduit. FIG. 4 shows a cross-sectional view of the optical transmission body. FIG. 5 shows an apparatus suitable for illuminating TLC plates with polychromatic light. FIG. 6 shows another device that can use polychromatic light. DESCRIPTION OF SYMBOLS 1... TLC plate, 2. Substance spot, 3... Monochromator, 5... Optical transmission body, 7... Photodiode, 16... Light receiver, 14. Polychromator, 20
...Receiver.

Claims (1)

[Scope of Claims] 1) A light source, means for forming a line image of light on the thin plate, means for effecting relative movement between the line of light and the thin plate, reflected by or by the thin plate; includes an optical unit l- for recording the emitted light and means for converting, storing and evaluating the analog signal obtained when recording its reflection or emitted light with a parameter proportional to the amount of matter; A device for quantitatively and automatically evaluating a thin layer chromatogram by recording with a densitometer the light reflected or emitted by the developed thin layer plate, the apparatus comprising: The optical unit for recording the emitted light includes a large number of optical sensors, and the area on the thin layer plate recorded by each sensor is such that the distribution according to the concentration side within this area can be considered uniform. These ξ
parameters can be stored in the data storage means and after all segments of the material spot on the laminar plate have been recorded, all transformed and stored parameters can be aggregated to observe the material in the laminar spot. An automatic quantitative evaluation device for thin layer chromatograms, characterized in that it comprises a converter capable of giving a value corresponding to a quantity, a storage means, a computer and a readout unit. 2) The device according to claim 1, wherein the optical sensor is arranged in a line parallel to the line image of the light on the thin layer plate. '5) About 5 to about 1. 3. The device according to claim 2, wherein the individual sensors of the IJOO are arranged in a line. 4) The device according to claim 2, wherein 5 to 50 optical transmission bodies are arranged in a line, each of which is connected to a photodiode. 5) Apparatus according to claim 2, in which a linear array having about 50 to 1,000 elements is used as an optical sensor. 6) Device according to claim 1, in which a polychromatic light source is used and the light reflected or emitted by the laminar plate is directed to the optical sensor by means of a vorichromator. 7) A device according to any one of claims 2 to 6, in which the sensor line is arranged movably to receive different wavelengths. 8) The device according to claim 6, which uses a two-dimensional multi-element optical receiver for receiving different wavelengths simultaneously. 9) If the device is configured as a multi-wavelength device, at least one standard wavelength and one measuring wavelength, in each case a two-dimensional multi-element reception R%, a line or line assigned to a specific wavelength: 9. The device according to claim 7 or 8, which performs simultaneous measurements using different optical sensors. 10) Automatic quantitative evaluation of a thin layer chromatogram by recording the light reflected or emitted by the developed thin layer plate with a densitometer, characterized by using the apparatus according to claim 1. Method.