You are required to prepare two plates:
- D-Ribose, D-Glucose, D-Fructose, D-Galactose.
- D-Glucose, D-Galactose, Maltose, Lactose.
You are provided with 5% solutions of Ribose, Glucose, Fructose, Galactose, Lactose and Maltose in water.
The chromatogram is developed by placing in a covered beaker containing less than 1cm of butan-1-ol:acetone:phosphate buffer (pH 5) in the ratio (40:50:10). Allow the solvent to run to within about 1cm of the top of the plate (see later) and then remove the plate from the beaker and allow to dry in a fume cupboard. The fumes are not particularly harmful, but they can be unpleasant when large numbers of students are working in the laboratory.
The samples are visualised by dipping the plates in a mixture of anisaldehyde with 0.5% concentrated sulphuric acid. (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
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. Glucose is a Hexose sugar so has 6 carbon atoms and forms a pyranose ring this is why it has a lower Rf value than that of ribose which is a smaller molecule.
Fructose: had an Rf value of 0.4 this probably due to it having a similar shape to ribose (see structure), it has 6 carbon atoms so is bigger than ribose which is why it dose not have the same Rf value as ribose.
Galactose: had an Rf value of 0.28 on plate1 and 0.26 on plate 2 these are similar values and the difference is probably due to not finding the exact centre of the spot on the two different plates. Galactose has a very similar structure to that of glucose it has 6 carbons and is a hexose sugar it also forms a pyranose ring the only difference is that on carbon 4 the OH group is above the ring and not below as in glucose. 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 α or a β 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 β 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.
References
1, Indge,B; Rowland,M; Baker,M, A New Introduction To Biology, Hodder & Stoughton 2000
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