1) A mixture of unknown amino acids can be separated and identified by means of paper chromatography. The position of the amino acids in the chromatogram can be detected by spraying with ninhydrin, which reacts with amino acids to yield highly coloured products (purple).
The filter paper, which contains a thin film of water trapped on it, forms the stationary phase. The solvent is called the mobile phase or eluant. The solvent moves up a piece of filter paper by capillary action. As the solvent moves past the simple spot of dye, there is a competition between the ability of dyes to attach to the adsorbed water, and the ability of dyes to dissolve in the solvent
This is a type of partition between the stationary phase (the adsorbed water) and the mobile phase (the solvent). As different dyes have different partition between the mobile and the stationary phases they would be carried forward to different extents. Dyes which are more soluble in the solvent than in water are carried further up by the mobile phase.
Fresh solvent is continuously moving up and there is, in fact an enormous number of successive solvent extractions occur. At a suitable time, the experiment is stopped by removing the filter paper from the solvent. The retardation factor Rf, can be calculated.
Rf = distance traveled by spot/distance traveled by the solvent.
The Rf value of any substance may be about the same whenever we use that particular solvent at a given temperature. However, the Rf value of a substance differs in different solvents and at different temperatures.
When the chromatographic paper has fully dried, outline the spots, mark the centers of each of the spots, and note their colors. (Not all amino acids give the same color with ninhydrin). Measure and record the distances the solvent and each of the amino acids traveled from the origin. Use these distances to calculate Rf values for each sample.
Comparison of the spots should enable you to identify the amino acid(s) present in your unknown sample.
2) In paper chromatography support material consists of a layer of cellulose highly saturated with water. In this method a thick filter paper comprised the support, and water drops settled in its pores made up the stationary “liquid phase.” Mobile phase consists of an appropriate fluid placed in a developing tank. Paper chromatography is a “liquid-liquid” chromatography.
3) Some solvents cannot be used at variable concentrations since they are not miscible with water.
Chloroform, Tetrachloromethane and Butanol, cannot be diluted with water to form variable concentrations. If water, an alcohol and chloroform (or tetrachloromethane) are mixed together, an upper layer of water and a bottom layer of alcohol + chloroform is formed. This shows that alcohol prefers to dissolve in chloroform rather than in water.
4) Infrared spectroscopy is one of the most useful spectroscopic techniques in organic chemistry because it is a rapid and effective method for identifying the presence or absence of simple functional groups within a compound. When IR energy is passed through a sample, absorption bands are observed and these bands are then correlated with types of chemicals bonds which can provide important information about the nature of functional groups in the sample. An IR spectrum has energy measured as frequency of wavelength against intensity of the absorption. This is the result of molecular vibrations. The atoms making up a molecule are in constant motion and the movements of the atoms relative to each other can be described as vibration. The photons of IR radiation absorbed by an organic compound have just the right amount of energy to stretch or bend its covalent bond. An absorption band appears in an infrared spectrum as a result of this vibration. These bands are affected by different vibration modes of the compound, its polarity, as well as its bond order. There are different types of vibration modes at a tetrahedral carbon. They are stretching, bending, scissoring, rocking, twisting, and wagging.