Continuous Development Thin-Layer Chromatography
In contrast to conventional thin-layer chromatography,which is carried out in a closed tank,the continuous development or continuous flow technique allows the upper end of the plate to project through a slot in the cover of the developing chamber.When the developing solvent reaches the slot,continuous evaporation occurs,producing a steady flow of solvent over the plate.In conventional thin-layer chromatography,spot migration ceases when the solvent reaches the top of the plate,after which the spots simply enlarge by diffusion.In the continuous flow process,spot migration continues as long as the plate remains in the tank and the developing solvent is not exhausted.
Development may be continued for several hours after the solvent reaches the top of the plate,to provide adequate migration of the spots.Usually spots of a standard solution,a test solution,and a mixture of equal amounts of test and standard solutions,are initially applied at a standard distance from the base of the plate.Identity of the standard and test substances is confirmed by their migrating equal distances from the origin and by the observation that the two substances applied as a mixture show no tendency to separate.
Amajor advantage of continuous development thin-layer chromatography stems from the greater solvent selectivity for solvents of low solvent strength.Solvent strength refers to the property of a developing solvent that causes solutes to migrate,and it is strongly influenced by the polarity of the solvent.Increasing the solvent strength by adding a more polar solvent causes the RFvalue to increase.Solvent selectivity refers to the ability of a solvent system to produce different RFvalues for closely related substances.In conventional thin-layer chromatography,a solvent system giving an RFvalue in the range of 0.3to 0.7,but with adequate selectivity to permit separation of the substances being examined is usually selected.It is much easier to find solvent systems producing adequate migration than to find those affording adequate selectivity.
Solvent systems of lower strength generally exhibit higher selectivity,but are difficult to employ in conventional thin-layer chromatography because they result in very little migration before the solvent reaches the top of the plate.Migration may be increased,however,by repeated drying and redevelopment of the plate or,more conveniently,by providing means for evaporation of solvent at the top of the plate,which results in continuous development.Two techniques are used:continuous development and short-bed continuous development thin-layer chromatography.
An RFvalue cannot be measured in continuous development thin-layer chromatography.Substances may be compared either by their migration distance over a fixed period of time or by comparison with the migration of a standard substance applied to the plate.
CONTINUOUS DEVELOPMENT
Apparatus— Acceptable apparatus and materials for continuous development thin-layer chromatography are the same as those described under conventional Thin-Layer Chromatography,except as follows.
Adeveloping chamber is used that consists of a rectangular tank,approximately 23cm ×23cm ×9cm,equipped with a glass solvent trough and a platform about 3.75cm high to elevate the solvent trough above the base of the tank.The chamber is fitted with a cover having a 21-×6-cm slot in the front edge.
Procedure— Apply the standard solution,the test solution,and a mixture of equal amounts of the standard solution and the test solution to a line about 2cm from the base of the plate.Place the plate in the elevated empty solvent trough with the adsorbent on the underside of the leaning plate.The adsorbent rests against a piece of heavy (about 1mm thick)2filter paper measuring 20cm ×3cm,folded lengthwise and placed over the front edge of the tank.Place the developing solvent in the trough;set the cover in place,and seal all openings except where the adsorbent contacts the paper wick.The plate extends about 1cm beyond the top of the tank.After the solvent reaches the top of the plate,allow development to continue for an appropriate time.Then remove and dry the plate,and detect the spots by suitable means.
SHORT-BED CONTINUOUS DEVELOPMENT
Amajor advantage of the short-bed technique derives from the fact that solvent velocity is inversely related to bed length.Since spot migration depends upon the total amount of solvent passing over the plate,the short-bed permits useful migration to be obtained in a reasonable time with solvent having very low solvent strength.Lower diffusion in solvents of low solvent strength produces smaller and more dense spots,which enhances both detectability and discernment of small differences in migration distance.
Apparatus— Acceptable apparatus and materials for short-bed continuous development thin-layer chromatography are the same as those described under conventional Thin-Layer Chromatography,except as follows.
Ashallow developing chamber3approximately 22cm ×9cm ×3cm,equipped with a cover plate and tight-fitting polytef wings that enable the chamber to be sealed against the plate,is used.The inside bottom of the chamber contains ridges that support the plate and allow it to be inserted at different angles,thereby varying the length of the plate contained within the tank.
Procedure— Apply the standard solution,the test solution,and a mixture of equal parts of the standard solution and the test solution to a line about 2cm from the base of the plate.Place the plate in the developing chamber (adsorbent side up),and add the developing solvent to the chamber.No paper wick is employed.After the solvent reaches the top of the plate,allow development to continue for an appropriate time.Then remove and dry the plate,and detect the spots by suitable means.