The chromatography paper is made of cellulose, a polar substance, and the compounds within the mixture travel farther if they are non-polar. More polar substances bond with the cellulose paper more quickly, and therefore do not travel as far. Chromatography is used for the separation of a mixture of compounds into its individual components. There are several ways to accomplish this but one thing that all chromatography shares in common is the use of a mobile phase and a stationary phase. What other mixtures can be separated in this way? My favorite use of paper chromatography is to separate the pigments found in leaves. A green pigmented leaf can easily be separated to show that it contains chlorophyll-a, chlorophyll-b, xanthophyll and carotene. The final chromatogram would look like this: Two way chromatography has completely separated out the mixture into four distinct spots. If you want to identify the spots in the mixture, you obviously can't do it with comparison substances on the same chromatogram as we looked at earlier with the pens or amino acids examples. Chromatography is used to separate and identify all sorts of substances in police work. Drugs ranging from narcotics to aspirin can be identified in urine and blood samples, often with the aid of chromatography.
You are making the assumption that if you have two spots in the final chromatogram which are the same colour and have travelled the same distance up the paper, they are most likely the same compound. There is no need to measure the Rf values because you can easily compare the spots in the mixture with those of the known amino acids - both from their positions and their colours. The paper is then stood in a suitable solvent and left to develop as before. In the diagram, the mixture is M, and the known amino acids are labelled 1 to 5.