It is also called thin layer chromatography in EnglishThin layer chromatography，ChromatographyIt is an important experimental technology for rapid separation and qualitative analysis of a small amount of substances. It belongs to solid-liquidAdsorption chromatography, both column chromatography andPaper chromatographyOn the one hand, it is suitable for the separation of a small amount of samples (a few micrograms, even 0.01 micrograms);On the other hand, when making thin plateAdsorption layerThickened and enlarged, therefore, it can also be used to refine samples. This method is especially suitable for low volatility or high temperature, which is easy to change and cannot be usedGas chromatographic analysisSubstances.It is an analytical method of chromatographic separation on a thin layer (usually about 0.25mm thick) formed by evenly coating adsorbent, carrier or other active substances on a flat plate (such as glass plate, plastic sheet, metal sheet, etc.).
Place the sample solution on one end of the thin-layer plate with a capillary tube, place it in a closed tank, and add a suitable solvent as the mobile phase.Due to the capillary principle, the solvent is absorbed, moves along the plate, and drives the components in the sample to move forward. This process is called unfolding.Due to the different properties of each component and the different moving distance, the component spots separated from each other can be obtained after a certain distance is expanded.Appropriate methods can be used to display the position of each component on the board. If the component itself has color, it can be directly observed. Otherwise, it can be determined by spraying color reagent or observing fluorescence under ultraviolet light
Thin layer chromatography of Scutellaria baicalensis
Thin layer chromatography of Scutellaria baicalensis
Spot position.Usually usedRatio shift valueRf refers to the characteristic of component movement, which is defined as:
Rf=component moving distance/solvent front moving distance
=The distance from the origin to the center of the component spot/the distance from the origin to the front edge of the solvent.
In thin layersolventUnder constant experimental conditions such as temperature, Rf value of each substance is constant, and it does not change with the change of solvent moving distance.Rf vspartition coefficientK's relationship:
Rf=1/(1+αK)，
α is a constant determined by the properties of thin layers.It can be seen that the larger the K value is,soluteAssigned tostationary phaseThe greater the trend of Rf, the smaller the value of Rf;On the contrary, the smaller the value of K, the smaller the value ofmobile phaseThe greater the trend of Rf, the more important the Rf value is for qualitative analysis.staymobile phaseWhen the stationary phase and development conditions are fixed, Rf value is the characteristic of the component, which can be used for qualitative identification.In practice, samples and known pure substances are often developed at the same time to compare their movement and conduct qualitative analysis.
quantitative method
Announce
edit
It can be divided into elution assay, thin-layer chromatography and in-situ scanning method:
Elution assay
① Elution determination method: scrape off the adsorbent at the developed component spot, elute and dissolve the component with appropriate solvent, and then conduct quantitative determination with appropriate method.The common methods are colorimetry, ultraviolet visible spectrophotometry, and can also be usedElectrochemical analysisEtc.
In situ scanning
② In situ scanning method, place the expanded thin plate on theOptical densitometerIt is irradiated with light of a certain wavelength, and the spot is moved across the optical path at the same time. Because the spot absorbs light, the peak shape can be drawncurveThe content is calculated by comparing the peak area with the absorption of the standard sample.
Thin layer chromatography
Adsorption chromatography is the most commonly used thin-layer chromatography.Common adsorbents are silica gel, alumina, etc.Like other chromatography methods, thin-layer chromatography can also be used for distributionIon exchange, molecular exclusion and other mechanisms, that is, the material used to pave the thin layer is correspondingly replaced withstationary phaseCarrierIon exchanger, gel, etc. Its operation is basically the same as the adsorption method.
Plate makingPavingThin-layer plateThe base plate shall be clean and flat, and can be paved by dry method or wet method.Currently, the wet method is commonly used to make boards, that is, to mix the adsorbent and adhesive (such as calcined gypsum) in a certain proportion, add proper amount of water to mix evenly, and then slowly move the homogenate through the base plate with a coater, place it to dry, and then use it after proper baking and activation.If the adsorbent is directly and evenly spread into a thin layer without adhesive and water, it is a dry process board.At present, there are all kinds of prepared thin slabs on the market, collectively referred to as precast slabs.
openThere are many ways, and the above line method is the most commonly used.Place the thin-layer plate vertically or obliquely, and add the developing solvent to the lower part to make it move from bottom to top.The downward rule is to use filter paper to lead the solvent to the upper end of the thin layer to make it flow from top to bottom.When developing in parallel, lay the plate flat, and the solvent is sucked up to the end of the thin-layer plate where there is a sample, and then develop.When using a round thin plate, place the sample atcenter of a circleNear, make the solvent from the center of the circlecircumferenceDirection movement, called circular expansion or radial expansion;Place the sample point at the circumferential position so that the solvent moves from the circumference to the center of the circle is developed centripetally, which is suitable for the separation of components with large f values.Scrape off the adsorbent near the point sample, so that the solvent can only be developed through the narrow part near the sample point, so the solvent front edge is in an arc development mode, also known as radial development, which may be better for components that are difficult to separate.
After developing once, take out the thin layer plate to volatilize the solvent, and then use the same solvent or another solvent to develop in this direction for several times.Place the sample point at one corner of the square thin plate, first unfold it in one direction, then rotate the plate 90 °, and then unfold it in another direction in two directions.Multiple unfolding and two-way unfolding can enhance the separation effect.
Features Simple operation, simple equipment, exceptOptical densitometerIn addition, no special equipment is needed, the separation effect is good, and the time is short. Many samples can be separated on one plate at the same time.Except for low boiling point substances, various inorganic and organic compounds can be separated.
Application and development Widely used in oilchemical industry, medicine, biochemistry, etc.The sample dosage is usually several to several hundred micrograms, which is a practical and effective micro separation and analysis method.This method can also be used to separate and prepare a large number of samples, that is, use a larger and thicker thin layer plate to dot the sample solution into strips at the starting point, so that milligram samples can be separated.
High performance liquid chromatographyIt is a method for efficient separation of fine particles, and has also been used for thin layer chromatography.Good separation effect can be obtained by using 5~10 μ m adsorbent to make plates. In order to distinguish from general thin-layer chromatography, it is called high performance thin-layer chromatography.Its advantages are small sample amount (only nanogram sample), small development distance and short development time. It is an important development of thin-layer chromatography.
