General Introduction
Chapter 1 Overview
Chapter 2 Equipments and Operation
Chapter 3 Main factors infiuencing the performance of thin-layer chromatography
2.1.1 Thin-layer plate：Commercial pre-coated plates（conventional plate and high performance plate）are usually used to obtain chromatograms of better resolution and reproducibility。However，the quality of precoated plates from different manufacturers varies due to processing differences and the use of inconsistent grades of the raw materials（e.g. silica gel，adhesive materials，etc.）。For instance，some commercial plates are suitable for development of lipophilic constituents while not suitable for hydrophilic substances which often need water to be added into the mobile phase。Thus，the quality and reproducibility of the chromatography obtained were greatly affected，which made the use of high quality pre-coated plates important especially in the case of samples containing complex components。Therefore，the selection of plates is a priority。
Chromatograms obtained by plates from both aboard and domestic sources as well as self-coated are compared to gain impressions on the difference of plates and the proper one is chosen。For the TCM tested in this atlas，imported commercial conventional plates and high performance plates （MACHEREY-NAGEL Co. Ltd.，Germany）are preferred for their better quality and affordable cost。
2.1.2 Sample application equipment：The most common and convenient instrument for sample application is the quantitative sampling capillary （or micro-cap），with capacities of 0.5μl，1.0μl，2.0μl，5.0μl，10.0μl， etc.The semi-automatic or automatic band-wise spray sampling mode is adopted in this atlas to ensure best chromatogram quality。There are a few examples where spot-wise sample application is used。In the absence of more sophisticated automatic or semi-automatic sampling equipment，manually contact punctuated sample application can also be used。 For this sampling mode，careful operation is essential for prevent solvent diffusion。This is especially important when a larger volume of sample is applied onto the plate。
2.1.3 Developing chamber（TLC tank，or TLC chamber）：There are two types ofTLC developing glass chambers：horizontal and vertical。The latter is further subdivided into fiat-bottom chamber and twin trough chamber。 The twin trough chamber is recommended in this atlas for its advantages of solvent saving，easy preequilibrium and controllable relative humidity when developing。For most TCM samples，it is suitable to use vertical unsaturated or partially saturated development in which solvent vapor is diffused and equilibrated in developing chamber for some time before the thin layer plate is inserted for development。If a saturated development environment is needed，filter paper of the same size as the inner lateral of the chamber is placed within the inner surface of the developing chamber to achieve the saturated condition as quickly as possible。
2.1.4 Derivatization and detective equipment：A developed chromatogram can be seen directly as a colorful image if the analytes appear as color bands/spots in daylight。However，most of the components in crude drugs are not visible under white light，needing a derivatization agent to enhance visibility。For example，volatile oils in many crude drugs can only be visible on the TLC plate when a chemical reagent such as vanillin-sulfuric acid solution is added。Applications of the derivatization agent include spraying method and immersion。 Pressurized aerosol bottles containing reagent solution can spray a fine uniform mist onto the surface of the plate。This spraying method is most commonly used。The immersion method requires a special glass immersion tank。 The developed thin layer plate is placed into the immersion tank steadily and vertically for one or more seconds。The residual agents on the back of the glass plate need to be wiped clean when it is removed out from the immersion tank。In some cases，the plate may require heating after derivatization for bands/spots visualization in daylight or under ultraviolet light。
The ultraviolet lamp cabinet for observing TLC images is equipped with two lamps of long wavelength（365 nm）and short wavelength（254 nm）。The former is used for observation of fluorescent chromatogram，while the latter is usually used to observe fluorescence quenching dark bands/spots under fluorescent background on silica GF2s4 plate。 The power of fluorescent lamp and filter specifications should be considered when selecting the ultraviolet lamp。
