Activity No. 4 CHROMATOGRAPHY Casiño, Jeanne Pearl C. Bachelor of Science in Chemistry Ms. Nova Fe E. Anorico, RCh, MSc April 2021 INTRODUCTION 1.1 Rationale of the Experiment Chromatography is a technique used to separate components in a mixture and can be used to partially identify the components. It works in a way were each liquid undergoes adsorption in a slightly different way and spends more or less time in either the solid or the liquid phase. One of the liquids might spend much longer in the solid phase than in the liquid, so it would travel more slowly over the solid; another one might spend less time in the solid and more in the liquid, so it would go a bit faster (Woodford, 2020). In this experiment, we used two different types of chromatography in separating the organic components, the thin layer chromatography and column chromatography. Thin layer chromatography is a technique used to isolate non-volatile mixtures, the process involved the use of the most known adsorbent, the silica gel. The mixture was placed in a plate and is emerged in a bath of solvent up until below the mark. The components started to separate and were then gradually moving upward, from the mobile phase to stationary phase with different velocities. To calculate the retention factor, the following formula was used: Rf = Distance Travelled by Sample Distance Travelled by Solvent Column chromatography on the other hand, is another solid-liquid technique in which the two phases are a solid (stationary phase) and a liquid (moving phase). The theory of column chromatography is analogous to that of thin-layer chromatography. The sample is dissolved in a small quantity of solvent (the eluent) and applied to the top of the column (Welleslly, n.d.). During the laboratory instead of the solution rising up, it flows down through the column filled with the adsorbent. The column was loaded with mixture, sand, solution and solvent. When everything is already in tacked and settled, the data was the collected through a numerous labeled flask by opening the stopcock. Along with it, the component started to separate differentiating which is which with colors of red, blue and such. Just like the tlc, this method establishes its own equilibrium between the solute adsorbed on the silica gel and the eluting solvent that is flowing down through the column. Objectives of the Experiment The main objectives of this experiment is to separate organic compound through chromatography. The experiment also addressed the following: 1. To separate the components using thin layer chromatography. 2. To separate the product using column chromatography. METHODOLOGY 1. Experimental Set-Up Part A. Separating Components using Thin Layer Chromatography Figure 1. Thin Layer Chromatography Experiment 1.1 Preparation of Materials and Chemicals In the thin layer chromatography experiment, a specific materials and chemicals were prepared. The experiment required the following: tlc plate, solution with components, solvent, pencil, uv light, solvent, developing chamber, beaker cover, capillary tube and flask. 1.2 Procedure in Separating the Compound using TLC To perform the experiment, a line was drawn near at the bottom of the tlc plate with the use of a pencil. A capillary tube was then inserted in a flask containing the component, to suck some of the solution through a capillary action. The content in the tube was disposed right unto the line in the tlc plate by pressing the end of the tube three times. The spotted tlc plate was put inside the beaker but was reassured that the line was above the solvent to avoid washing out the component. For the solvent not to evaporate the beaker was then covered. The solvent began to rise from the mobile phase to the stationary phase of the plate, wherein the component is also dragged upward. The plate was then removed when the solvent almost reaches the top of the plate. With the use of a pencil, the place where the solvent stop was mark with a line. The plate was then leave for a moment to dry. Since the component isn’t visible enough an ultraviolet light inside a special box was used to trace and analyzed the data. 1.3 Schematic Diagram Figure 2. Schematic Diagram for Separating the Components using TLC TLC Plate and the Sample Compound - mark a line near at the bottom of the plate - Insert a capillary tube in a flask to suck some of the components in the solution - Deposit the content in the tube to the mark of the plate Developing Chamber - Put the plate inside the chamber Add a solvent and make sure It will not reach the mark Close the beaker with something to avoid evaporation Mobile Phase to Stationary Phase - - Watch the solvent rise from MP to SP Notice the component being drag upward and separates Remove the plate as soon as it reaches nearly at the top and mark the spot Use a uv light carefully to gather the data needed End of Experiment PART B. Separating the Compound using Column Chromatography Figure 3. Column Chromatography Experiment 1.4 Preparation of Materials and Chemicals In the column chromatography experiment, a specific materials and chemicals were prepared. The experiment required the following: Liquid funnel, glass column, eluent, sand, adsorbent, mixture, flask, iron stand, iron lamp, cotton, stopcock, rotary evaporator, tlc and pipette. 1.5 Setting-Up the Compound and the Column A cotton was placed at the bottom of the column using a wire. The column was then clamp in an iron stand. A layer of sand was added inside together with the solvent filling one-third of the column. After that, an amount of silica gel powder was poured into the beaker containing the solvent, inside the fume hood to avoid inhaling the toxic. The mixture was then stirred until its completely slurry. To get in to the column a spatula and solvent was used to push the slurry inside and gently patted to settle. Another set of sand was added onto the column along with the solvent to drip. 1.6 Loading the Column with Mixture The mixture was dissolve in the smallest amount as possible for it to be pipetted onto the column. It was then drip directly into the sand without touching the sides of the column. Finally, small amount of solvent was drain to load the mixtures onto the stationary phase. 1.7 Adding the Solvent A solvent was added to the column through a pipette by squirting it to the side, until it reaches several inches above the sand. 1.8 Collecting Fractions The solvent was dripped out of the column and was collected through a series of labeled flask. It was noticed that the components began to separate as they move through the column. To separate properly an optical flow rate was applied. To complete the process, an amount of eluent was gradually added onto the column until all the data needed were collected. 1.9 Analyzing Fraction A 5-10 vertical lines were drawn in the plate, and was numbered according to its corresponding fractions. Each line has a spot for each fractions and the plates were then developed. All the fractions were then combine into a rotary evaporator to evaporate the solvent away. 2.0 Schematic Diagram Figure 2. Schematic Diagram for Separating the Compound using Column Chromatography Setting-Up - Place a cotton at the bottom Clamp the column Add sand and solvent Mix solvent and silica powder Put the slurry in the column using Spatula and settle Loading Column with Mixture and Solvent - Dissolve the mixture into the smallest amount and drip into the column using pipette - Drain the solvent until mixture reaches SP - Add solvent through the side Collecting and Analyzing Fraction - Drip the solvent Collect the data needed Analyze End of Experiment RESULTS AND ANALYSIS The following data shown in Table 1.1 and Table 1.2 are obtained from the results, after completely separating the components using two different types of chromatography. The thin layer chromatography and column chromatography. Table 1.1 Results in Separating the Components Using Thin Layer Chromatography Result Identification Rf values Separated Non-polar 0.91 Separated Polar 0.45 Solution and Solvent Table 1.1 shows the result in separating the components using tlc. The solution containing the components was placed in a plate containing silica gel and put in a solvent bath. With this, the eluting power of the solvent will increase making those polar and non-polar substance dissolved and eluted. In this case, you will see that both components are completely separated but one is a polar and the other is non-polar. Looking at the retention factor non-polar seems to travel a farther distance than the polar, that is because polar molecules have stronger bonds which is ideal for adsorbent. It will spend most of its time in the stationary phase that slower its movement, while as of the less polar it was carried out quickly up unto the mobile phase (Chromatography, 2007). Table 1.2 Results in Separating the Product using Column Chromatography Result Entire Sample Product Separated Table 1.2 shows the result in separating the product using column chromatography. The column was filled with the entire sample according to their respective layers, wherein the product must sit in between the sand and the slurry also known as the stationary phase. The solvent level was lowered, in order for the separating process to run smoothly. Solvent plays a vital role in separating the product, because of its polarity. When it passes through the column it affects the relative rates in which the mixture moves through( ). So as for the entire sample after it was settled inside the column and ready for the process, they are being collected through series of flasks, and an amount of solvent was gradually being added for those polar molecules will compete the polar solvent. In analyzing if the product was successfully separated the entire data was run through a rotary evaporator. Rotary evaporator is a device used to remove solvents from the sample by evaporation (“Rotary evaporator”, 2020). The solvent began evaporating until the product was completely isolated. REFERENCES Love Food Love Science. (nd). Chromatography Experiment. Retrieved from https://www.ifst.org/lovefoodlovescience/resources/chromatographyexperiment#:~:text=Chromatography%20is%20a%20technique%20used,to%20partially%20identify%20t he%20components.&text=There%20are%20two%20phases%20involved,phase'%2C%20the%20salt%20s olution. Woodford, C. (2020). Chromatography. Retrieved from https://www.explainthatstuff.com/chromatography.html CHEMISTRY LibreText. (2019). Thin Layer Chromatography. Retrieved from https://chem.libretexts.org/Ancillary_Materials/Demos_Techniques_and_Experiments/General_ Lab_Techniques/Thin_Layer_Chromatography BYJU’S. (nd). Thin Layer Chromatography. Retrieved from https://byjus.com/chemistry/thinlayerchromatography/#:~:text=Thin%20Layer%20Chromatography%20is%20a,%2C%20cellulose%2 C%20or%20silica%20gel. Chem 344 TLC. (nd). Thin Layer Chromatography. Retrieved from https://www2.chem.wisc.edu/deptfiles/OrgLab/handouts/CHEM%20344%20TLC%20info.pdf Columbia University. (2007). Chromatography. Retrieved from www.columbia.edu/cu/chemistry/ugrad/hssp/EXP_8.html Wikipedia contributors. (2020). Rotary evaporator. Retrieved https://en.m.wikipedia.org/wiki/Rotary_evaporator#:~:text=A%20rotary%20evaporator%20 from
