Abstract Paper Chromatography uses a chromatogram paper as its stationary phase and the solvent as the mobile phase. Retention factor is the ratio of the distance travelled by the sample to the distance travelled by the solvent. This experiment aims to separate organic compounds, to compute Rf values and to identify unknown compounds using Rf values. The ten samples underwent paper chromatography to determine the components of the unknown sample. The mobile phase allowed the compounds to be moved. In identifying the sample, the color, number of spots and Rf values were observed.
Introduction Chromatography is a separation technique based on the partition between two phases, mobile and stationary. This technique involves a sample to be dissolve in a solvent (mobile phase) then forced into the stationary mobile. From the two phases, the sample would travel along the stationary phase with the mobile phase until such time that the sample is dissolved. Chromatography is divided into several types. Column Chromatography uses a powder adsorbent placed in a vertical glass column as its stationary phase and its solvent as the mobile phase.
The mixture is distributed as the solvent moves down the column. Thin Layer Chromatography, or TLC, also uses a powder absorbent which is fixed to a metal, plastic or glass plate as its stationary phase. The mixture to be analyzed is placed near the bottom of the plate and it is then submerged to a specific amount of solvent which is the mobile phase. Gas Chromatography requires a long metal or glass column containing a high boiling liquid which would serve as the stationary phase and an inert gas to flow on the column as the mobile phase.
Lastly, Paper Chromatography uses a cellulose chromatogram paper as its stationary phase and the solvent as the mobile phase. In 1903, chromatography was first discovered by Mikhail Tswett, a Russian botanist, by producing a colorful separation of plant pigments through calcium carbonate. From the discovery, column adsorption chromatography was coined. Archer Porter Martin won 1952 Nobel Prize for discovering Paper Chromatography. Fritz Cremer successfully separated carbon dioxide and oxygen by gas chromatography.
Retention factor (Rf) is the ratio of the distance travelled by the sample to the distance travelled by the solvent, multiplied by 100 (Eq. 1). Rf is used to comparing the results in order to identify an unknown sample. Impurities and interactions made by a sample to another would affect Rf value. Equation 1-Retention Factor This laboratory technique has been helpful in various ways. It is used by pharmaceutical companies in preparing large amounts of pure compounds. In the food industry, chromatography is used to separate preservatives and additives to ensure food quality.
It is also used to identify drug intake in the urine. This experiment used paper chromatography to separate organic compounds, to compute Rf values and to identify unknown compounds using Rf values. In this experiment, the organic compounds to be used are in Table 1. Table 1-Test Compounds Organic Compounds (Dyes) Methyl Orange Source: http://commons. wikimedia. org/wiki/File:Methyl-orange-2D-skeletal. png Methylene Blue Source: http://commons. wikimedia. org/wiki/File:Methylene_blue. svg Fluorescein Source: http://commons. wikimedia. org/wiki/File:Fluorescein. png Indigo Carmine Source: http://en. wikipedia. org/wiki/Indigo_carmine Erythrosine Source: http://en. wikipedia. org/wiki/Erythrosine Methodology All the chemicals used were prepared by the laboratory assistant before the experiment had started. The materials needed were 600 mL beaker, capillary tubes, petridish or watch glass and chromatogram paper. The retention factor (Rf) of each test compounds was determined by paper chromatography process (Process 1). Figure 1- Chromatogram Paper Table 2-Order of Dyes Spot No. Dyes 1 MO 2 MO + MB 3 MB 4 F + MB 5 F 6 I + MB 7 I 8 E + I 9 E 10 Un Legend: MO – Methyl Orange F – Fluorescein E – Erythrosine MB – Methylene Blue I – Indigo Carmine Un – Unknown Figure 2-Chromatogram Paper with Dyes Process 1 Results and Discussion Ethanol had reached 60 mm in the chromatogram paper after 15 minutes. Based from the results, methyl orange travelled the most in paper while methylene blue travelled the least (Figure 3). Figure 3 – Results The Retention factor (Rf) tells how far the dye would travel depending on the distance travelled by the solvent (Equation 1). It ranges from 0 to 1.
Since it is impossible that the solvent wouldn’t be absorbed by the paper, the dye hadn’t travelled with the solvent if Rf is zero and the dye would infinitely travel with the solvent if Rf is equal to 1. The group’s unknown showed three dyes, fluorescein, methyl orange and methylene blue. These dyes were compared to the color and distance travelled by the pure dyes. Table 3-Distance travelled by the sample Sample Dyes Distance travelled MO 48 mm MO + MB 48 mm 8 mm MB 8 mm F + MB 53 mm 8 mm F 53 mm I + MB 45 mm 8 mm I 45 mm E + I 50 mm 45 mm E 50 mm Un 8 mm 48 mm
53 mm Table 4-Retention Factor Dyes Rf MO 80 MO + MB 80 13 MB 13 F + MB 88 13 F 88 I + MB 75 13 I 75 E + I 83 75 E 83 Un 13 80 88 The distances travelled by the samples listed in table 3 were the values used in computing for the retention factor (Rf). Here, the distances were measured in millimeters, then it was divided by the distance travelled by the solvent which is 60 mm. To get Rf values that are whole numbers, it was multiplied by 100. Table 4 shows the calculated Rf values of the samples and the unknown. The solubility of the dyes had affected their Rf values.
If the dyes are more soluble to ethanol, then it would obtain a high Rf value. From table, Methylene Blue had the lowest Rf since it is less soluble. Ethanol is a polar solvent while Methylene Blue is a non-polar. On the other hand, Methyl Orange, Fluorescein, Indigo Carmine and Erythrosine are polar compounds which gave them high Rf values. Polar solvents are much considered than the non-polar. This is due to the attraction between the cellulose fiber and sample. If the samples are polar, it would tend to react with the paper than the non-polar solvent. This would create a partition chromatography.
Some precautions were observed during the experiments to avoid erroneous results. The paper must be handled at the edges so that the spots wouldn’t get infected by the fingers. The beaker must be covered tightly. The reason for covering the beaker is to make sure that the atmosphere in the beaker is saturated with solvent vapor. To help this, the beaker is often lined with some filter paper soaked in solvent. Saturating the atmosphere in the beaker with vapor stops the solvent from evaporating as it rises up the plate. The sample spot must be above the solvent level to avoid mixing them with the liquid to be absorbed.
In stapling the edges of the paper, it must not overlap so that the mobile phase may not travel in a path that is not vertical. The sample spots must not be to heavy or light to get an accurate value of Rf and they must be separated to each other so that the dyes wouldn’t be mixed. Do not move the beaker, because moving the beaker will affect the results from the paper. After the paper is removed from the beaker, mark immediately the colored regions because it may be nearly invisible after the solvent evaporates. There were some incorrect results found in the experiment due to some sources of errors.
The light pencil line drawn in the paper wasn’t straight. The sizes of the spots were varying that might have affected the movement. The concentrations of each compound weren’t considered. The solvent wasn’t adsorbed equally by the chromatogram paper. Conclusion and Recommendation The organic compounds were successfully separated using paper chromatography. This was made possible by the mobile phase, solvent which allowed the compounds to be moved. The distance travelled was dependent on the solubility of the sample. The more soluble the sample is, the farther it would travel.
The retention factor is the quotient of distances travelled by the sample and by the solvent. The Rf is multiplied by 100 just to obtain a result that is a whole number. Rf values are used to compare with other samples. These are comparable to their solubility in accordance with the solvent used in chromatography. Paper chromatography is useful to identify unknown samples such as organic compounds. Mixtures are separated into its components due to their difference in solubility. To identify them accurately, their Rf values are compared to the pure form of the compounds.
They are also compared qualitatively through their colors. The number of spots created in the chromatography determines the number of the compounds present. The researchers recommend drawing a pigment line rather than a pigment spot. When separated, pigments will appear as bands rather than smears so the calculations can be made easier.