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Spotting is the main source of quantitative error in TLC. Experiments have shown that quantitative capillaries are more suitable for smaller volume spotting; microinjectors are more suitable for larger volume spotting. This is mainly because the micro-syringe is greatly affected by small bubbles and solution back crawling. In order to avoid spotting errors between different quantitative capillaries, it is recommended to use the same quantitative capillary on a thin plate. However, care should be taken to replace the sample with ultrasonic or different polar solvents. When preparing the sample, the viscosity of the solvent should not be too high to facilitate spotting; if the boiling point of the solvent is too low, the injection volume is apt to change, and too high will change the composition of the developing agent; if the solubility of the sample is too high, the sample will be hollow. Commonly used solvents are methanol, ethanol, and acetone. The origin of the classical TLC sample is generally 3 mm in diameter and 1 - 2 cm in pitch. The bottom margin is 1.5 cm. The origin of the HPLC sample is generally 1 mm in diameter and 5 mm in pitch. The bottom margin is 1 cm.
2 , the choice of developing agent TLC compared with HPLC, a prominent advantage is the choice of mobile phase for greater flexibility. The purpose of the mobile phase is chosen such that the RF value of most samples is between 0.1 and 0.7 and a good separation is achieved, corresponding to the proper strength and composition of the mobile phase. The greater the strength of the mobile phase, the larger the solute RF value, but it is likely to reduce the separation ability; in addition, it is difficult to separate the more complex mixture by a single solvent, and the multi-solvent system is used according to the principle of similar compatibility. The general developer system is selected as follows: According to the nature of the separated sample, the properties of the thin-layer chromatography plate, a binary solvent system is selected, and the solvent ratio is adjusted to find a suitable RF value. After the suitable RF value is found, the binary with the same polarity parameter is sought. Two solvent systems, with three components forming a triangle, can be seen: the vertices of the triangle are binary systems, the edges are ternary systems, the ternary system is within the triangle, and the polarity is consistent, according to geometry The principle draws the composition of any point. This method is more intuitive and simpler.
3 , TLC's universal color rendering method Ideal color development, high sensitivity, stable spot color, good contrast between spots and background, the size of the spot and the depth of the color are proportional to the amount of matter. The general color rendering method appears to be particularly important where the sample composition is not fully known. The general color method mainly includes:
(1), ultraviolet irradiation method: convenient, does not destroy the sample;
(2), iodine vapor method: strong versatility, combined with the ultraviolet method sensitivity is higher than the two methods used alone;
(3) Fluorescent reagent: The fluorescent background is made, so that no fluorescent substance is identified in the original ultraviolet light, and the fluorescent substance is more obvious;
(4), sulfuric acid solution: effective for most organic substances, but destructive.
Use of thin layer chromatography and precautions
1. How to improve the efficiency of spotting?