Paper Chromatography Pascual, Maria Elena E. Abad Cruz Landiza Tambong NASC 2115 BSIOP 4-1 Group 1 Mr.

Dennis Macapagal DEPARTMENT OF NATURAL SCIENCES COLLEGE OF SCIENCE POLYTECHNIC UNIVERSITY OF THE PHILIPPINES Retardation Factor In chromatography, the retardation factor (Rf) is defined as the fraction of an analyte in the mobile phase of a chromatographic system. In planar chromatography in particular, the retardation factor (Rf) is defined as the ratio of the distance traveled by the center of a spot to the distance traveled by the solvent front. Ideally, the values for RF are equivalent to the R values used in column chromatography. It is important to note that, although the term retention factor is sometimes used synonymously with retardation factor (Rf), in regards to planar chromatography, the term is not defined in this context. However, in column chromatography, the retention factor (k) is defined as the ratio of time an analyte is retained in the stationary phase to the time it is retained in the mobile phase.

METHODOLOGY

Prepare 5o% each of ethanol and water by mixing 25ml each. Pour about 15ml of the solution in the developing chamber and cover.

Make the filter paper rectangular. Along one side, about 1cm from the edge, 4 tracks are marked by pencil. Label each with the code of the food color. Spot each of the food color about 3mm. And also the unknown. Wait until dry.

Once the spots are dry, place it in the developing chamber.

Remove the paper and quickly outline the location of each spot. Compute the Rf values and compare

Results and Discussion 1. Food Color/s Dye Red Yellow Indigo Green Solvent Distance (cm) 4.6 4.6 4.5 4.5 6.0 Rf .77 .77 .75 .75

Each of the food color moved up as the solvent raised in the filter paper. The farthest distance was gained by color red and yellow.

2. Unknown Solution Trial 1 2 3 Solvent Red 4.4 4.4 4.5 6.0 Rf .73 .73 .75 Blue 5.6 5.7 5.8 Rf .93 .95 .97 Yellow 4.5 4.5 4.6 Rf .75 .75 .77

The color of the unknown solution separated into yellow, red, and blue and moved up as the solvent raised in the filter paper. The farthest distance was gained by color blue in trial 3.

POST LAB QUESTIONS: 1. What is paper chromatography? What is the principle involved in this separation technique? Paper chromatography is one method for testing the purity of compounds and identifying substances. Paper chromatography is a useful technique because it is relatively quick and requires small quantities of material. Separations in paper chromatography involve the same principles as those in thin layer chromatography. In paper chromatography, like thin layer chromatography, substances are distributed between a stationary phase and a mobile phase. The stationary phase is usually a piece of high quality filter paper. The mobile phase is a developing solution that travels up the stationary phase, carrying the samples with it. Components of the sample will separate readily according to how strongly they adsorb on the stationary phase versus how readily they dissolve in the mobile phase. When a colored chemical sample is placed on a filter paper, the colors separate from the sample by placing one end of the paper in a solvent. The solvent diffuses up the paper, dissolving the various molecules in the sample according to the polarities of the molecules and the solvent. If the sample contains more than one color, that means it must have more than one kind of molecule. Because of the different chemical structures of each kind of molecule, the chances are very high that each molecule will have at least a slightly different polarity, giving each molecule a different solubility in the solvent. The unequal solubilities cause the various color molecules to leave solution at different places as the solvent continues to move up the paper. The more soluble a molecule is, the higher it will migrate up the paper. If a chemical is very nonpolar it will not dissolve at all in a very polar solvent. This is the same for a very polar chemical and a very nonpolar solvent. 2. Which one is the mobile phase? Stationary phase? The stationary phase is usually a piece of high quality filter paper. The mobile phase is a developing solution that travels up the stationary phase, carrying the samples with it. 3. What are the pros and cons of this technique? Advantages of Paper Chromatography: This analytical method is quick to perform and easy to master. With a correctly chosen mobile phase (chromatographic solvent), an analyst can rapidly determine the number of constituents of a mixture sample. Sometimes, paper chromatography even allows one to positively identify these constituents. Disadvantages of Paper Chromatography: Like all analytical methods, paper chromatography has its limitations. Some mixtures are very difficult to separate by paper chromatography; and any species that is not coloured is difficult to observe on the chromatogram. Also, paper chromatography is solely an analytical method, not a preparative one. Because the sample size is so small, it is difficult to perform further analysis after the sample's contents have been chromatographically separated. This is in contrast to methods such as column chromatography, which are frequently use to

preparatively separate larger amounts of mixtures. Lastly, paper chromatography can only be used in qualitative analysis. It is not possible to extract meaningful information about the quantitative content of a mixture from a paper chromatogram. 4. What are the considerations in choosing a chromatographic solvent? Non-polar molecules in the mixture that you are trying to separate will have little attraction for the water molecules attached to the cellulose, and so will spend most of their time dissolved in the moving solvent. Molecules like this will therefore travel a long way up the paper carried by the solvent. They will have relatively high Rf values. On the other hand, polar molecules will have a high attraction for the water molecules and much less for the non-polar solvent. They will therefore tend to dissolve in the thin layer of water around the cellulose fibres much more than in the moving solvent. If water works as the mobile phase as well being the stationary phase, there has to be some quite different mechanism at work - and that must be equally true for other polar solvents like the alcohols, for example. Partition only happens between solvents which don't mix with each other. Polar solvents like the small alcohols do mix with water. 5. If the substance to be analyzed is colorless, how should one visualize the results? In some cases, it may be possible to make the spots visible by reacting them with something which produces a coloured product. A good example of this is in chromatograms produced from amino acid mixtures. Suppose you had a mixture of amino acids and wanted to find out which particular amino acids the mixture contained. A small drop of a solution of the mixture is placed on the base line of the paper, and similar small spots of the known amino acids are placed alongside it. The paper is then stood in a suitable solvent and left to develop as before.