THIN-LAYER CHROMATOGRAPHY
In thin-layer chromatography,the adsorbent is a relatively thin,uniform layer of dry,finely powdered material applied to a glass,plastic,or metal sheet or plate,glass plates being most commonly employed.The coated plate can be considered an “open chromatographic column”and the separations achieved may be based upon adsorption,partition,or a combination of both effects,depending on the particular type of support,its preparation,and its use with different solvents.Thin-layer chromatography on ion-exchange films can be used for the fractionation of polar compounds.Presumptive identification can be effected by observation of spots of identical RFvalue and about equal magnitude obtained,respectively,with an unknown and a reference sample chromatographed on the same plate.Avisual comparison of the size of the spots may serve for semiquantitative estimation.Quantitative measurements are possible by means of densitometry,fluorescence,and fluorescence quenching;or the spots may be carefully removed from the plate,followed by elution with a suitable solvent and spectrophotometric measurement.For two-dimensional thin-layer chromatography,the chromatographed plate is turned at a right angle and again chromatographed,usually in another chamber equilibrated with a different solvent system. Apparatus— Acceptable apparatus and materials for thin-layer chromatography consist of the following. Flat glass plates of convenient size,typically 20cm ×20cm.1 An aligning trayor a flat surface upon which to align and rest the plates during the application of the adsorbent. Astorage rackto hold the prepared plates during drying and transportation.The rack holding the plates should be kept in a desiccator or be capable of being sealed in order to protect the plates from the environment after removal from the drying oven. The adsorbentconsists of finely divided adsorbent materials,normally 5to 40µm in diameter,suitable for chromatography.It can be applied directly to the glass plate or can be bonded to the plate by means of plaster of paris (hydrated calcium sulfate)[at a ratio of 5%to 15%]or with starch paste or other binders.The former will not yield as hard a surface as will the starch,but it is not affected by strongly oxidizing spray reagents.The adsorbent may contain fluorescing material to aid in the visualization of spots that absorb ultraviolet light. Aspreader,which,when moved over the glass plate,will apply a uniform layer of adsorbent of desired thickness over the entire surface of the plate. Adeveloping chamber that can accommodate one or more plates and can be properly closed and sealed as described under Ascending Chromatography.The chamber is fitted with a plate-support rack that supports the plates,back to back,with the lid of the chamber in place. Atemplate(generally made of plastic)to aid in placing the test spots at definite intervals,to mark distances as needed,and to aid in labeling the plates. Agraduated micropipetcapable of delivering 10-µLvolumes.Total volumes of test and standard solutions are specified in the individual monograph. Areagent sprayerthat will emit a fine spray and will not itself be attacked by the reagent. An ultraviolet light source suitable for observations with short (254nm)and long (360nm)UVwavelengths. Procedure— [NOTE—In this procedure,use purified water that is obtained by distillation.]Clean the plates scrupulously,as by immersion in chromic acid cleansing mixture,rinsing them with copious quantities of water until the water runs off the plates without leaving any visible water or oily spots,then dry.It is important that the plates be completely free from lint and dust when the adsorbent is applied. Arrange the plate or plates on the aligning tray,place a 5-×20-cm plate adjacent to the front edge of the first square plate and another 5-×20-cm plate adjacent to the rear edge of the last square,and secure all of the plates so that they will not slip during the application of the adsorbent.Position the spreader on the end plate opposite the raised end of the aligning tray.Mix 1part of adsorbent with 2parts of water (or in the ratio suggested by the supplier)by shaking vigorously for 30seconds in a glass-stoppered conical flask,and transfer the slurry to the spreader.Usually 30g of adsorbent and 60mLof water are sufficient for five 20-×20-cm plates.Complete the application of adsorbents using plaster of paris binder within 2minutes of addition of the water,since thereafter the mixture begins to harden.Draw the spreader smoothly over the plates toward the raised end of the aligning tray,and remove the spreader when it is on the end plate next to the raised end of the aligning tray.(Wash away all traces of adsorbent from the spreader immediately after use.)Allow the plates to remain undisturbed for 5minutes,then transfer the square plates,layer side up,to the storage rack,and dry at 105for 30minutes.Preferably place the rack at an angle in the drying oven to prevent the condensation of moisture on the back sides of plates in the rack.When the plates are dry,allow them to cool to room temperature,and inspect the uniformity of the distribution and the texture of the adsorbent layer;transmitted light will show uniformity of distribution,and reflected light will show uniformity of texture.Store the satisfactory plates over silica gel in a suitable chamber. Place two filter-paper wicks,18cm in height and as wide as the length of the developing chamber,into the chamber,add about 100mLof the solvent (sufficient to have a depth of 5to 10mm at the bottom of the chamber),seal the cover to the top of the chamber,and allow the system to equilibrate;it is essential that the wicks become completely wet.Alternatively,the chamber may be completely lined with filter paper.In either case,assure that the filter paper dips into the solvent at the bottom of the chamber.Where vapor saturation of the chamber by these methods is undesirable,it is so indicated in the individual monograph. Apply the Test solution and the Standard solution,as directed in the individual monograph,at points about 1.5cm apart and about 2cm from the lower edge of the plate (the lower edge is the first part over which the spreader moved in the application of the adsorbent layer),and allow to dry.Avoid physical disturbance of the adsorbent during the spotting procedure (by the pipet or other applicator)or when handling the plates.The template will aid in determining the spot points and the 10-to 15-cm distance through which the solvent front should pass. Place a mark 10to 15cm above the spot point.Arrange the plate on the supporting rack (test spots toward the bottom),and introduce the rack into the developing chamber.Allow the solvent in the chamber to reach the lower edge of the adsorbent,but do not allow the spot points to be immersed.Put the cover in place,and maintain the system until the solvent ascends to a point 10to 15cm above the initial spots,this usually requires about 15minutes to 1hour.Remove the plate from the developing chamber,mark the solvent front,air-dry the plates,and observe first under short-wavelength UVlight (254nm)and then under long-wavelength UVlight (360nm).Measure and record the distance of each spot from the point of origin,and indicate for each spot the wavelength under which it was observed.Determine the RFvalues for the principal spots (see Glossary of Symbols).If further directed,spray the spots with the reagent specified,observe,and compare the test chromatogram with the standard chromatogram.