Column and thin layer chromatography of plant pigments
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Noel Angelo P. Kalacas*, Hanna Mae Laluces, Ina Bianca Lanuza Department of Chemistry, College of Science
*Corresponding author; e-mail: knight_BeNcH66@yahoo.com
Chromatography is a powerful technique for separating and/or identifying the components in a mixture. There are different types of chromatography and each has its own strengths and weaknesses. In this experiment, pigments of the chili pepper were extracted with the use of hexane, hexane-DCM, DCM, and DCM with methanol; then, the extract was introduced into the column and eluate were collected, this process is the column chromatography (CC). The purity of the components was determined by using the thin layer chromatography (TLC). Ultraviolet spectroscopy and iodine staining were used to visualize the developed TLC plate and the Retention Factor was measured. Keywords: Chromatography, column chromatography, TLC and visualizing agent, elution, capsicum frutescens
Chromatography can be defined as the separation of a mixture, in which the mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. Chromatography was first developed by the Russian botanist Mikhail Tswett in 1903 as he produced a colorful separation of plant pigments through a column of calcium carbonate. The principle behind chromatography is that different substances have different partition coefficient between mobile and stationary phases, and consequently each will move through a system at a different rate, resulting in complete separations. There are various types of chromatography, depending on the physical states of the phases. Employing a gas, the mobile phase is termed as gas chromatography (GC) or vapor phase chromatography (VPC). Separations using gas chromatography involve vapor phase versus adsorption and/or equilibria. Liquid chromatography (LC) refers to any chromatographic process that employs a mobile liquid phase. Chromatographic separations can also be carried out using thin layer chromatography (TLC) and column chromatography (CC) which a variety of supports, including immobilized silica on glass plates. Chromatography separates a substance into its component parts, which is very useful, as substances are often unique in their composition. It can identify a substance and show how it differs from others that may look alike on the surface. All types of chromatography are useful for analytical purposes. Under appropriate conditions, all types of chromatography can be used for preparative scale separations. In every type of chromatography there are three elements to be considered: the size of the sample (load), relative separation of components (resolution), and the speed. It would be ideal if all three elements could be maximized so that the complete separation of samples of any desired size could be quickly achieved. In practice, generally two of these elements can be maximized at the expense of the third. For routine analytical work, resolution and speed are maximized at the expense of the load. In preparative scale separations, load and speed can be maximized but then separations are usually incomplete. Complete separations of large samples can be achieved but the overall operation is likely to be slow and tedious, and may involve the use of large quantities of solvent that must be distilled for reuse, or discarded. In the experiment, Column Chromatography and Thin Chromatography were used.
Figure 1. Column Chromatography
Column chromatography is advantageous over most other chromatographic techniques because it can be used in both analytical and preparative applications. Not only it can be used to determine the number of components in a mixture, but it can also be used to separate and purify considerable quantities of those components for subsequent analysis. This...
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