Column and Thin Layer Chromatography: the Separation of Spinach Pigment

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Column and Thin Layer Chromatography: The Separation of Spinach Pigment

Abstract
Spinach extract was separated into fractions containing compounds of similar polarity by column chromatography. Based on solid-liquid phase partitioning, this separation technique exploited the different polarity of the compounds in the spinach extract. Three fractions with different colors were obtained. The extract and its fractions were analyzed using thin layer chromatography (TLC). The TLC results showed that there was one compound (Rf=0.979) in the first fraction; there were three compounds (Rf1=0.839, Rf2= 0.691, Rf3=0.149) in the second fraction; there was one compound (Rf=0.017) in the third fraction. The separations of compounds which went to the first and third fraction were relatively satisfying, while the second fraction had several kinds of compounds. Introduction

Chromatography is the separation of compounds or ions by distribution between two phases—a mobile phase and a stationary phase. The technique is based on the differential absorptivities of the constituents between these two phases, due to different properties of the compounds to be separated and the nature of the two phases involved. If one constituent adheres more to the stationary phase than the mobile one, separation will be achieved.

There are several different types of chromatography, such as thin layer chromatography (TLC), gas chromatography (GC) and column chromatography. All the chromatographic methods are based on partitioning of molecules between a stationary phase and a mobile phase. In order to measure the amount of partitioning between the two phases, each compound has an unique Partition Coefficient (Kp), which is defined as the ratio of concentrations of the compound between the stationary phase and the mobile phase: Kp=[x]sp/[x]mp. Therefore, if Kp is greater than 1, the substance adheres more to the stationary phase; if Kp is smaller than 1, the substance adheres...
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