Content1. What is thin layer chromatography machine?
1.1 What is thin layer chromatography?
1.1.1 Basic principle of thin-layer chromatography
1.1.2 Classification of Thin Layer Chromatography2. Classification of thin layer chromatography machine
2.1 Conventional thin layer chromatography scanner
2.2 Thin-layer digital imaging analyzer3. Use of thin layer chromatography machine
4. How to buy thin layer chromatography machine?
What is thin layer chromatography machine?
Thin layer chromatography machine is a kind of lab scanner for quantitative analysis of specimens using the principle of thin layer chromatography.
What is thin layer chromatography?
is named because the separation is performed on a flat, thin layer. Thin layer chromatography
is a chromatographic separation technique for the separation, identification and quantification of mixed samples by using a suitable solvent as the mobile phase and a support coated on a support plate as the stationary phase. It is a fast, simple and microscopic chromatographic method developed in the late 1940s.
In 1938, Izmailor and Schraiber coated a thin layer of alumina on a microscope slide, spotted the substance to be separated on the thin plate, and placed the unfolding agent vertically on the sample point ZX with a capillary tube, and the unfolding agent flowed out from the capillary tube, thus successfully separating the components of a variety of plant alcohol extracts.
In the 1950s, K. Rehner and Miller, based on the previous research, used silica gel as the adsorbent and gypsum as the binder, coated the silica gel on a glass plate to make a thin layer, and carried out the separation of substances, which is also the thin layer chromatography
in the modern sense.
Basic principle of thin-layer chromatography
takes advantage of the different adsorption capacity of each component to the same adsorbent so that in the process of mobile phase (solvent) flowing through the stationary phase (adsorbent), adsorption, desorption, re-adsorption and resorption are continuously generated, thus achieving the separation of each component from each other.
Adsorption is an important property of surfaces. Any two phases can form a surface, and adsorption is the intensive phenomenon of the substance of one of the phases or the solute dissolved in it on this surface. Adsorption can occur on surfaces between solids and gases, between solids and liquids, and between adsorbed liquids and gases.
The adsorption process is reversible, and the adsorbed material can be desorbed under certain conditions. A dynamic equilibrium can be established between the molecules adsorbed on a certain surface area of the adsorbent and the molecules leaving this surface in the same unit of time, called adsorption equilibrium. The adsorption chromatography process is a dynamic equilibrium process that constantly produces equilibrium and disequilibrium, adsorption and desorption.
The reason why molecules of a substance can stay on the surface of a solid is that the molecules (ions or atoms) on the surface of the solid and the molecules inside the solid are not equally attracted to each other. Inside the solid, the forces interacting between molecules are symmetrical and their force fields cancel each other out. The forces on the molecules at the surface of the solid are asymmetric, with the inward side being subjected to a large force from the molecules inside the solid and the surface layer being subjected to a small force, and thus the gas or solute molecules are attracted to stay when they encounter the surface of the solid in motion by this residual force.
The main principle of using silica gel and alumina as support agents, for example, is the difference between the adsorption force and the partition coefficient, which allows the mixture to be separated. As the solvent moves along the adsorbent, it takes the components of the sample with it, while continuous adsorption and desorption, as well as repeated partitioning, takes place.
Classification of Thin Layer Chromatography
Thin-layer chromatography can be divided into distribution thin-layer method, adsorption thin-layer method and ion exchange thin-layer method according to the nature of the stationary phase and different separation mechanisms. Among them, the adsorption thin-layer method and distribution thin-layer method are most widely used.
A. Dispersion thin-layer method
Distribution thin-layer method uses a liquid as the stationary phase. The liquid stationary phase is pre-attached to a carrier (a porous chemically inert solid substance), then the stationary phase is coated on the plate, and the sample is unfolded.
B. Adsorption thin layer method
The process of uniformly spreading the stationary phase (adsorbent) on a smooth surface of a thin layer plate and spotting the sample solution to be analyzed on the appropriate position at one end of the plate is called spotting.
The sample solution point is called the origin point. Put the thin plate in a closed unfolding tank (chamber), dip the sample end in the appropriate official solvent, with the capillary action of the adsorbent on the thin plate, the solvent carrying the separated components to move forward is called development.
During development, the components in the sample are continuously adsorbed, desorbed, resorbed and desorbed between the stationary phase and the developing solvent. Due to the different adsorption capacity of different components of the fixed phase, the components that are easy to be adsorbed move relatively slowly, while the components that are difficult to be adsorbed move relatively quickly. After a period of time, when the unfolding agent front reaches a predetermined position, the thin layer plate is removed, and the components with different adsorption power can form spots separated from each other on the thin layer plate. If the components are colorless substances, they can be positioned after color development by physical or chemical methods.
C. Ion exchange thin layer method
The ion exchange thin layer method uses ion exchange resin as the stationary phase. Usually, the ion exchange resin is crushed into 200-40O mesh, and then coated on the surface of glass, metal sheet or other materials to make a thin layer of plate, and then point sample unfolding.
The components of the substance under test are exchanged with the ion exchange resin. The components with strong exchange capacity are exchanged first, and the components with poor exchange capacity are carried away by the unfolding agent and then exchanged again, so that the different components are separated.
Classification of thin layer chromatography machine
Conventional thin layer chromatography scanner
Conventional thin layer chromatography scanner is a full wavelength scanner that offers a choice of wavelengths in the range of 200-800nm and determines the substance content by detecting the intensity of light absorption by the sample. The scanner can also detect the intensity of fluorescence generated by UV irradiation at 254nm or 365nm, allowing for specific detection.
The scanning methods of conventional scanners are divided into single-beam scanning, dual-beam scanning and dual-wavelength scanning.
A. Single beam scanner
Single-beam scanning uses a single beam. The instrument is simple in structure, but the baseline is unstable and rarely used in practice.
B. Dual beam scanner
The dual-beam scanner uses two beams of the same wavelength to scan simultaneously, with one beam scanning the sample unfolding channel and the other beam scanning the blank area next to the sample channel, so that the blank absorption can be deducted, partially eliminating the error caused by uneven spreading in the thin-layer plate unfolding direction. However, it cannot eliminate the error caused by uneven spreading in the spreading direction.
C. Dual wavelength scanner
Two beams of different wavelengths alternately scan the unfolded channel area of the sample. One wavelength should be set at the wavelength selection for the position of maximum absorption of the sample and the other for the position of minimal absorption. This method can basically eliminate the error generated by uneven spreading of the plate, so the scan baseline is very stable.
Thin-layer digital imaging analyzer
Thin-layer digital imaging analysis is a thin-layer analysis technique that uses digital imaging equipment to obtain information on the light intensity of each point on a thin-layer plate and then analyzes the obtained image. There are two types of digital imaging devices, namely cameras and scanners. Since the digital scanner uses progressive imaging technology, the digital scanner is referred to as a progressive scanner for the purpose of distinguishing the point-by-point scanning of a conventional thin-layer scanner.
Like conventional thin-layer scanners, both cameras or progressive scanners have light detection systems, which are components that linearly convert the amount of light into an electrical signal. The difference is that cameras and progressive scanners can further convert the electrical signal into a computerized image, where the color shades of individual dots (pixels) in the image reflect the intensity of the light.
Therefore, thin-layer digital imaging is closer to human eye observation and detection, and the results are more intuitive, making it ideal for identification, especially for fingerprint mapping of herbal medicines.
Use of thin layer chromatography machine
a. Mix 1 part of stationary phase and 3 parts of water in a mortar in one direction. After removing the air bubbles on the surface, pour it into the applicator. Move the applicator smoothly on the glass plate for coating (thickness 0.2-0.3mm). Remove the thin layer of coated glass plate, place it on the horizontal table to dry at room temperature, and then bake it at 110℃ for 30 minutes. Place in a desiccant oven and set aside. Check the uniformity before use (it can be examined by transmitted light and reflected light).
b. The sample was spotted on the thin layer plate with a spotter, generally as a round spot. The sample baseline is 2.0cm from the bottom edge, the diameter of the sample points and the distance between the points are the same as the paper chromatography method, and the distance between the points can be seen as the spread of spots to not affect the detection. Point sample must be careful not to damage the surface of the thin layer.
c. If you need to saturate the cylinder with spreading agent in advance, add enough spreading agent to the cylinder and stick two strips of filter paper on the wall with the same height and width as the cylinder, dip one end into the spreading agent, seal the cover of the top of the cylinder and make the system balanced or operate according to the text. Put the sample into the development agent of the development cylinder, and immerse the sample into the development agent to a depth of 0.5~1.0cm from the bottom edge of the plate (do not immerse the sample into the development agent). Seal the cylinder cover. Remove the laminate after development to the specified distance (generally 10-15cm) and dry it. Then test according to the provisions under each species.
d. If the chromatographic spots need to be scanned for detection by a thin layer chromatography machine or quantified by scanning the chromatographic spots directly on a thin layer, the thin-layer scanning method is used.
How to buy thin layer chromatography machine?
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