Thin Layer Chromatography Lab Report
- Dishita Sai Eluri
- Jun 10
- 10 min read
Woodbridge Academy Magnet School Dr. Wiamer Chemistry with Lab, DCC - Block 4A 25 September 2024
Purpose: The purpose of this TLC chromatography lab is to quantitatively determine the Rf values of the ink components of different pens and to qualitatively determine the unknown pens.
Procedure:
Materials required for the lab:
1. Safety Goggles
2. Circular filter paper (around 10 cm. in diameter)
3. 4 pens of the same color by different manufacturers
4. One unknown pen
5. One beaker (1000 mL)
6. Less than 100 mL of 70% isopropyl rubbing alcohol (isopropanol)
7. Parafilm
8. Pencil
9. Ruler
Lab Procedure:
1. Wear safety goggles and make sure long sleeves are pulled up and hair is tied back.
2. Acquire a circular filter paper and create a straight, horizontal line 1 inch above the edge of the paper.
3. Draw four different dots along the line using different pens while keeping track of the pen brands.
4. Draw a fifth dot with another pen to represent the unknown ink.
5. Pour a little less than 100 mL of isopropyl rubbing alcohol into a beaker.
6. Slightly curl the piece of filter paper and gently place the filter paper chromatogram in the isopropyl solvent.
7. The part of the filter paper with the line and dots drawn along it should be resting in the solvent.
Figure 1: How the Chromatogram should look when it is submerged in the solvent
8. Take a piece of parafilm and stretch it to fit the opening of the beaker, make sure not to tear it.
9. After covering the beaker with parafilm, wait for the solvent to travel up the chromatogram for about 40 minutes.
10. Take off the parafilm and dispose of it properly.
11. Slowly remove the chromatogram from the beaker.
12. With a pencil, trace a line along how far the solvent traveled, this is called the solvent line. Make sure to trace the line before the solvent evaporates from the paper.
13. Measure the distance the components of ink (D value) traveled and distance the solvent (L value) traveled for each dot to find the Rf values for all of the dots.
14. Calculate the Rf (Retention factor) values for each component of ink, including the unknown ink component.
15. Compare and contrast the Rf values and using the pen with the most similar ink components, find the identity of the unknown pen
16. Carefully dispose of the lab materials using the proper disposal procedures. 17.
Data:
Brands of Pens Used:
Dot 1 - Papermate
Dot 2 - Bic
Dot 3 - Tru Red
Dot 4 - Staples
Dot 5 - unknown (Bic)
Figure 2: The final chromatogram from the experiment
Table 1: L and D values (in inches)
Dot 1 - D-value | L - value | Dot 2 - D-value | L-value | Dot 3 - D-value | L-value | Dot 5 - D-value | L-value | Dot 5 - D-value | L-value |
1a = 0.21 | 1 | 2a = 0.9 | 1.1 | 3a = 0.95 | 1 | 4a = 0 | 1.1 | 5a = 0.5 | 1.75 |
1b = 0.5 | 1 | 5b = 1.5 | 1.75 | ||||||
1c = 0.85 | 1 |
Rf value Calculations:
Dot 1 | Dot 2 | Dot 3 | Dot 4 | Dot 5 |
1a. 0.21/1 = 0.21 in. | 2a. 0.9/1.1 = 0.81 | 3a. 0.95/1 = 0.95 | 4a. 0/1.1 = 0 | 5a. 0.5/1.75 = 0.29 |
1b. 0.5/1 = 0.5 | 5b. 1.5/1.75 = 0.86 | |||
1c. 0.85/1 = 0.85 |
Table 1: Rf values
Dot 1 | Rf value | Dot 2 | Rf value | Dot 3 | Rf value | Dot 4 | Rf value | Dot 5 | Rf value |
1a | 0.21 | 2a | 0.81 | 3a | 0.95 | 4a | 0 | 5a | 0.29 |
1b | 0.5 | 5b | 0.86 |
1c | 0.85 |
Discussion:
What is Chromatography?
Chromatography is a technique used to separate and identify components of a mixture and can be used for qualitative and quantitative analysis. This is possible because ions of different components move at different speeds. Components can be separated by their polarity because the polarity of the solvent affects the speed of the chromatography process. Molecules in the mixture go through the stationary phase, which is a solid or liquid phase coated on the surface of a solid phase. The next phase is the mobile phase, which flows over the stationary phase, is a gaseous or liquid phase. Combined, these two phases help separate the molecules.
Types of Chromatography:
There are several types of chromatography that are used to separate components in mixtures. Some of these types include Column chromatography, Ion-exchange chromatography, Gel permeation chromatography, Affinity chromatography, Paper chromatography, Thin Layer chromatography, Gas chromatography, Dye-ligand chromatography, Hydrophobic interaction chromatography, Pseudo Affinity chromatography, and High-pressure liquid chromatography.
Column Chromatography:
Column Chromatography uses columns to separate pure chemicals from a mixture. This can be done on a small scale or large industrial scale column. To prepare the column, a dry piece of glass or cotton is placed on the bottom of the column and sand is poured to neutralize the column base. This step is followed by the addition of silica and the solvent, which is also the eluent. The part of the mobile phase that transports the same components is known as the eluent. The eluent is added to the column, and then a solution of the organic material is pipetted onto the column walls. The movement of the compounds is based on the polarity of the molecules in the sample. After the eluent is added, the adsorption process, a process which transfers a molecule from a fluid to a solid surface, begins. If the component is non-polar, it will move faster towards the bottom of the column and enter the mobile phase. One by one, the components are separated by polarity until they are all in their own distinct test tubes at the bottom of the column.
Figure 3: Column Chromatography Diagram
Ion-exchange chromatography:
Ion-exchange chromatography separates compounds based on their net charge. In this type of chromatography, negatively or positively charged functional groups are covalently bound to a solid support, or a column. If the functional group is negatively charged, it yields an anion exchanger and if it is positively charged, it yields a cation exchanger. The charged components are adsorbed and then retained by an ion exchanger with the opposite charge. If the charge is negative, the compound is eluted from the column. Typically, anions move through the column faster. With this type of chromatography, scientists can determine the charge of the ions in the component sample.
Figure 4: Ion-exchange chromatography diagram
Gel Permeation chromatography:
In gel permeation chromatography (GPC), the separation of components depends on the size of the polymer molecules in the solution. In this type of chromatography, the mobile phase is a flowing liquid, and the stationary phase is a porous medium made of gel. The polymer is first dissolved into a solvent and then form coils. When introduced to the mobile phase, the bigger polymers cannot enter the pores of the gel medium, and they are eluted out of the mobile phase. The polymers of the correct size enter the pores on the stationary phase. As a result, the larger polymer coils exit the column more quickly. At the end of the lab, one would expect to see the polymers separated by size.
Figure 5: Gel permeation chromatography diagram
Affinity chromatography:
Affinity chromatography is the process of separating an immobilized ligand and its binding partner. Examples of this include an antigen and antibody or an enzyme and substrate binding. Affinity chromatography is useful in protein purification because it can use a protein’s naturally occurring biological structure for separation. First, a protein is purified and added to the
stationary phase containing the ligand, where it binds said ligand. The column is then washed to get rid of contaminants and the protein mixture is added to another column containing an affinity-tag sequence. Then, the affinity-purified samples can undergo buffer exchange to remove salts and prepare for downstream applications. The end result should have the binding proteins retained on the media, thus separating the proteins from the ligand.
Figure 6: Affinity chromatography diagram
Paper chromatography:
Paper chromatography is used to separate and analyze a mixture. In this type of chromatography, the paper is the stationary phase and the solvent is the mobile phase. Paper chromatography can be used to separate and identify a combination of amino acids. The paper is sprayed with ninhydrin, which causes a color change after interacting with the amino acids. The color mixture
is then separated using chromatography and capillary action that pulls the solvent up the length of the paper. In the end, the results show a distinct separation of color and the colored areas show the locations of the amino acids.
Figure 7: Paper chromatography diagram
Thin Layer Chromatography:
In thin layer chromatography, the stationary phase is usually a thin layer of silica gel on a piece of glass. The sample is added to one side of the TLC and placed in an organic chamber with the mobile phase. The solvent moves up the TLC using capillary action and causes the compound to move along the solvent front. The sample components move based on their affinities and then the TLC is taken out of the chamber. The separated components appear as dots on the TLC and can be measured to find their Rf values.
Figure 8: Thin Layer Chromatography diagram
Gas Chromatography:
Gas chromatography is applied to find out what specific compounds are in a mixture. To undergo this process, the components must be volatile. A sample of the substance is collected and prepared for gas chromatography. This sample will undergo the mobile phase of chromatography, in which an inert gas is inserted for the sample to react with. In this type of chromatography scientists can understand the polarity of different compounds and find their identities.
Figure 9: Gas chromatography diagram
Dye-ligand chromatography:
Dye-ligand chromatography is a very selective method to separate proteins from their ligands. Synthetic dyes act as substitute ligands for the proteins because of the chlorotriazine ring present in the dye. Dyes such as Cibacron Blue 3-GA can bind using ionic forces, hydrophobic reactions, or exclusion-diffusion. The dye is first purified and then immobilized onto the chromatography matrix. The chromatogram is run by loading the protein samples and then tested for the presence of the proteins.
Figure 9: Dye-Ligand Chromatography diagram
Hydrophobic Interaction Chromatography (HIC):
Hydrophobic interaction chromatography separates the proteins according to differences in their surface hydrophobic interactions. For this process, HIC uses a reversible interaction between the proteins and the hydrophobic ligand of a HIC resin. The proteins are bound to the HIC resin with a high salt concentration inside a binding buffer. When the ionic strength of the buffer is reduced, the interaction is reversed and the protein with the lowest hydrophobicity is eluted first. Using this process, proteins are separated based on their hydrophobic properties.
Figure 10: Hydrophobic Interaction Chromatography diagram
Pseudo-affinity chromatography:
Pseudo-affinity chromatography uses dyes as ligands to target and separate proteins. Since the dyes do not express high specificity, the dyes can capture a variety of proteins. First, the dye gets immobilized and then added to a gel with salt. The protein samples are added to the column and washed to remove any contaminants. At the end of this experiment, some of the proteins will stay inside the column and some will be eluted out, therefore separating the proteins from the dye.
Figure 11: Pseudo-affinity chromatography diagram:
High Performance Liquid Chromatography (HPLC):
HPLC allows the separation of compounds in a liquid sample. In this experiment, the mobile phase is delivered to the stationary phase, and then to a detector at a stable flow rate controlled by the solvent pump. A certain amount of the sample is injected into the column and then compounds inside the column are separated. The separated compounds are then detected by a detector and can be used for data analysis.
Figure 12: High Performance Liquid Chromatography (HPLC) diagram:
Formulas Used in the Lab:
In this specific TLC lab, one would use paper chromatography to separate ink components and measure their Rf (retention factor) values. The retention factor value represents how far the ink component traveled relative to the solvent front. For example, if the Rf value is 0.75, the ink component traveled 75% as far as the solvent front. The Rf value is calculated by the distance the component traveled, divided by the distance the solvent front traveled. In other words, the formula is D/L = Rf. If the Rf value is higher, it means the solute was more attracted to the solvent. Researchers can use this information to analyze quantitative data in relation to the chromatogram and use it for comparison.
Rf value formula:
Data Points from the Experiment:
Dot 1 (Papermate): The ink components traveled to three different spots. The Rf value for dot 1a is 0.21. This is because the ink traveled 0.21 inches and the solvent traveled 1 inch. 0.21/1 = 0.21, therefore the Rf value is 0.21. Similarly, for dots 1b and 1c, the ink traveled 0.5 inches for 1b (0.5/1 = 0.5), so the Rf value is 0.5, and for 1c, the ink traveled 0.85 inches, making the Rf value 0.85.
Dot 2 (Bic): The ink component of dot 2 traveled 0.9 inches, while the solvent traveled 1.1 inches. 0.9/1.1 = 0.81, therefore the Rf value is 0.81.
Dot 3 (Tru red): The ink component of dot 3 traveled 0.95 inches, while the solvent traveled 1 inch. Thus, the Rf value is 0.95 because 0.95/1 = 0.95.
Dot 4 (Staples): The ink component for dot 4 did not travel up the solvent front. This may be because the ink components stuck to the stationary phase, making the D value 0. The solvent traveled 1.1 inches, therefore the Rf value is 0 because 0/1.1 = 0.
Dot 5 (unknown - Bic): For dot 5, there were two spots where the ink components traveled. For dot 5a, the Rf value is 0.29 because the ink traveled 0.5 inches and the solvent traveled 1.75 inches. For dot 5b, the Rf value is 0.86 because the ink traveled 1.5 inches and the solvent
traveled 1.75 inches. Although the unknown pen yielded results, it must be noted that during the 45 minute period when the solvent traveled up the chromatogram, the chromatogram paper slipped and had to be readjusted inside the beaker.
Observations:
Observations are characteristics that are analyzed after the experiment. In this lab, as capillary action progressed and the solvent moved up the chromatogram, the ink components started to rise and change color. The changes in color from black pen included orange, dark blue, and a dark yellow color. There was no change in smell and no new chemical emerged from this lab. (See Figure 2.)
Figure 2: Final chromatogram from the lab
Results:
The Rf values that were calculated after the experiment allow for analysis and comparison of the ink components in all five pens. The unknown ink was most similar to dot 2a, which had a Rf value of 0.81, while dot 5b had a Rf value of 0.86. It must be noted however, that the chromatogram slipped inside the beaker and had to be readjusted. Overall, the results of this lab support that the unknown pen ink is from the Bic pen.
Conclusion:
In conclusion, this chromatography lab separated ink components using a stationary and mobile phase, allowing one to identify the identity of an unknown pen. The ink components were separated based on their adsorption and polar properties, allowing the researchers to analyze and compare and contrast data.
Advanced Study:
References:
Coskun, O. (2015, November 4). Separation techniques: Chromatography. Retrieved from ….https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206469/
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Column Chromatography - Principle, Procedure, Applications & Elution in Chromatography. ……(2020, June 3). Retrieved from https://byjus.com/chemistry/column-chromatography/
Coskun, O. (2015, November 4). Separation techniques: Chromatography. Retrieved from …..https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206469/
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Hydrophobic Interaction Chromatography - Cytiva. (n.d.). Retrieved from ….https://www.cytivalifesciences.com/en/us/solutions/protein-research/knowledge-center/protei ….n-purification-methods/hydrophobic-interaction-chromatography
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