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Chem Lab Report (Paper Chromatography)

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Daswani 6A (23) Experiment 20 Paper Chromatography Objective To illustrate the use of paper chromatography for the separation and identification of amino acids. Results Amino Acid Distance travelled /cm Rf value by solvent by amino acid Aspartic acid - alone - in mixture 5.3 5.3 1.2 1.2 0.2264 0.2264 Leucine - alone - in mixture 5.3 5.3 3.95 4.3 0.7453 0.8113 Lysine - alone - in mixture 5.3 5.3 3.1 3.3 0.5849 0.6226 Special Questions 1.> For each amino acid, compare your two Rf values with each other and with the specimen results Why do you think there is some variation? Both Rf values are the same for aspartic acid but there is some variation for leusine & lysine. This may due to: 1.> The grease on our fingers contaminated on chromatographic paper will affect greatly on the amino acids that are less polar. 2.> The measurement error of the distance travelled by solvent and/or amino acid. ...read more.


ammonia, water, etc. These will affect the polarity of the developing solvent and thus the Rf values. Precaution 1.> Small area with concentrated spot of amino acid should be applied to obtain a deeper colour of spots. 2.> The edges of chromatographic paper should not touch the sides of the beaker since he solution at edges will move faster due to capillary action. The spots will bend and Rf value is affected. 3.> The beaker should be saturated with the solvent before putting the chromatographic paper in. If the atmosphere is not saturated, there is a diffusion gradient between chromatographic paper and air, some solvent may vapourize from chromatogram and results in long tail of spots. 4.> The origin line should be marked by pencil, not ball pen, since the ball pen ink can dissolve in the developing solvent and the line fades gradually or even an additional spot can be observed. 5.> Ninhydrin solution should not spray too much on chromatogram since it will blur and spreads the coloured spots. ...read more.


Aspartic acid has the lowest solubility in mobile phase (most polar) than in stationary phase thus it moves slowest. After the mobile phase moved a certain distance, the spots (after drying) may be cut out and extracted with an appropriate solvent. If the solute is colourless such as amino acid, it can be located by spraying the chromatogram by ninhydrin solution to form a coloured spot. The colour of the complex formed between ninhydrin & aspartic acid is deep blue whereas that of leusine and lysine are purple. Other organic solutes may also be located by ultraviolet light (if it gives fluorescence in UV light) or by iodine vapour (I2 will dissolve in organic compound which results in a brown spot). 2-way chromatography (i.e. turn the chromatogram for 90� and obtain a second chromatography) may be applied for further chromatography to obtain a more distinct spot. If the paper medium is replaced with silica gel or alumina coated on a glass plate, it's called thin layer chromatography. In this case the separation is based on the effect of both adsorption and partition. ?? ?? ?? ?? ...read more.

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