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Chromatographic Separation of Carbohydrates.

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Chromatographic Separation of Carbohydrates Thin layer chromatography is a useful procedure, which can help in identifying carbohydrates. In this experiment Thin Layer Chromatography (TLC) will be used. All chromatographic systems have a stationary phase (in this case silica gel) and a mobile phase (in this case an organic mixture with some buffer). In this experiment you will separate two groups of four samples. You are asked to comment on the separation. The factors you should consider are structural i.e.: pentose, hexose, monosaccharide, and disaccharide. You will be using mini-plates, which only take about 20 minutes to develop. Method Using the side of the aluminium plate covered with silica gel, make a pencil mark about 1.5cm from the bottom of the plate, and label four sites to receive your samples. Do not handle the surface of the plate. The samples are applied using capillary tubes (one per sample). Allow the silica gel to draw out the sample by capillary action and try to keep your spots less than 5mm in diameter. ...read more.


(Now you know why you should not let the solvent run to the top of the plate). The plate is then heated in an oven until the colours develop. Measure the Rf values and try to relate these values to the structure. Results Plate 1 Solvent front 60mm Carbohydrate Distance moved (mm) Rf value Ribose 30 0.5 Glucose 20 0.33 Fructose 24 0.4 Galactose 17 0.28 Plate 2 Solvent front 66mm Carbohydrate Distance moved (mm) Rf value Glucose 24 0.36 Galactose 17 0.26 Maltose 9 0.14 Lactose 7 0.11 Discussion Structures: (1) Ribose: had the highest Rf value 0.5 as it is the smallest carbohydrate used, it is a pentose sugar and thus only has five carbon atoms. This allows it to move further through the plate in the time allowed than the others. Glucose: had an Rf value of 0.33 on plate 1 and 0.36 on plate 2 these are very similar and the slight difference may be caused by not finding the exact middle of the spot when measuring the distance it had moved. ...read more.


For these reasons I would expect it to have an Rf value similar to that of Glucose but slightly lower as it is less soluble than glucose. Maltose: had an Rf value of 0.14 this is due to maltose being a disaccharide so it is much larger than the other monosaccharides it is made of two glucose molecules joined with another glucose molecule by either an ? or a � 1,4 linkage. This means that the molecule did not move as far as its monosaccharide constituents. Lactose: had an Rf value of 0.11 this is due to it being a disaccharide so it is much larger than the other monosaccharides it is made of two hexose sugars glucose and galactose they are joined by a � 1,4, linkage it has a lower Rf value than that of maltose as the galactose monomer in lactose is less soluble than glucose which is the only monomer in maltose. Conclusion The experiment was successful and the individual spots were easily discernable and could be measured readily. The results for the duplicated spots such as D-Glucose and D-Galactose gave consistent results. ...read more.

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