The advantages and disadvantages of thin layer chromatography
(1) Define thin-layer chromatography, or thin-layer chromatography. Thin-layer chromatography is a chromatographic separation technique in which a support is uniformly coated on a support plate (commonly used glass plate, polyester cloth, etc.) to form a thin layer, and then expanded with a corresponding solvent. To separate, identify and quantify mixed samples.
(2) Classification Thin-layer chromatography can be divided into thin-layer adsorption chromatography (adsorbent), thin-layer distribution chromatography (cellulose), thin-layer ion exchange chromatography (ion exchange agent), thin-layer gel chromatography (molecular sieve gel), etc. In general experiments, thin-layer adsorption chromatography with adsorbent as the stationary phase is more commonly used.
(3) The advantages and disadvantages of the method
① Advantages: easy to operate, simple equipment, easy color development, etc., and fast deployment rate, generally only 15 to 20 minutes; the mixture is easy to separate, and the resolution is generally 10 to 100 times higher than that of previous paper chromatography. It is suitable for only
The sample separation of 0.01 μg can also separate samples larger than 500 mg for preparation, and corrosive color development agents such as concentrated sulfuric acid and concentrated hydrochloric acid can also be used.
Disadvantages: The separation effect of biopolymers is not ideal.
(3) Operation steps
① Preparation of thin-layer plates: Unless otherwise specified, 1 part of the stationary phase and 3 parts of water are ground and mixed in a mortar in one direction, after removing the bubbles on the surface, poured into the applicator, and smoothly moved on the glass plate. The applicator is coated (thickness is
0.2~ O.3mm), remove the coated thin glass plate, place it on a horizontal table to dry at room temperature, and then bake it at 110 ° C for 30 minutes, then place it in a drying oven with a desiccant for backup. Check its uniformity before use (can be viewed by transmitted light and reflected light).
② Spotting: Unless otherwise specified, use a spotter to spot on a thin-layer plate, usually dots. The baseline of the spotting is 2. Ocm away from the bottom edge. The diameter of the spotting points and the distance between the dots are the same as the paper chromatography method. The distance between the dots can be seen as the diffusion of the spots without affecting the detection. When spotting, care must be taken not to damage the surface of the thin layer.
③ Deployment: If the expansion chamber needs to be saturated with the expansion agent in advance, a sufficient amount of the expansion agent can be added to the chamber, and two filter paper strips as high and wide as the chamber can be pasted on the wall, one end is immersed in the expansion agent, and the lid of the top of the chamber is sealed to make the system balanced or operate according to the text. Put the thin layer plate of the sample into the development agent of the development chamber, and the depth of the immersion development agent is 0.5 to 1.Ocm from the bottom edge of the thin layer plate (do not immerse the sample point in the development agent). Seal the chamber cover, and wait for the expansion to a specified distance (usually 10-15cm). Take out the thin layer plate, dry it, and test it according to the regulations under each variety.
④ Color development: Take out the thin-layer plate and immediately spray the color developer to make it develop color, calculate the Rf value or make various colored spots appear, and compare it with the standard to determine its composition.
