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Thin Layer Chromatography Lab Report
Organic Chemistry (3201)
Austin Community College District
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Karina Santos June 16, 2020 Thin Layer Chromatography
I
Thin Layer Chromatography (TLC) is a laboratory technique used to identify different compounds present within a mixture. There are two phases: The stationary phase, which contains a solid and usually polar, and the mobile phase, which can be either a liquid or gas which can be a pure solvent or a mixture of solvents. 1 The TLC plates consist of a solid surface such as glass, metal or plastic with a thin adsorbent later on the surface and provides the stationary phase. In the lab, silica gel will be act as the solid, stationary phase and 99:1 ethyl acetate. The liquid, mobile phase will be represented by the use of acetic acid. If the compound in question forms dark spots on the TLC plate, then it will stick to the stationary phase because it is polar. They tend to form spots close to the starting, origin line of the TLC plate. On the other hand, the spots will flow with the mobile phase because the compound is less polar compared to the other compound. Nonpolar substances will also form spots that traveled a further distance compared to those that are slightly polar. A more polar compound will tend to strongly bind to the silica gel compared a compound less polar. To compare the distances between each substance, a retention factor (Rf) is calculated for each spot. It is determined by the ratio traveled by the compound divided to the distance the solvent has traveled. 1 An Rf value can be enlarged by raising the polarity of the solvent, while it can also be decreased by increasing the polarity of the compound. 2
II. Safety
Safety Data Sheet – NFPA3a-d Acetaminophen3a
Aspirin/ Acetylsalicylic Acid3b Dichloromethane
3c Ethyl Alcohol3d Health 2 1 2 3 Flammability 1 1 1 2 Conductivity 0 1 0 0 Other Hazards N/A 0 N/A 0 Table 1: Safety Data Sheet
III. Experiment 4 1. Obtain a TLC Plate of the appropriate size that can fit into the developing chamber.
- By using a ruler and pencil, lightly mark a line, called the Origin Line, one centimeter above the bottom of the TLC plate. Place markers 1 cm apart on the origin – there should be 4 markers. Mark each marker A-D.
- Spot the plate with a micropipette. First, spot the A and C markers with Standard X. Second*, spot the B and C markers with Standard Y. Finally*, spot the last marker, D, with the unknown solution.
- Add the pre-measured suitable solvent in the developing chamber. Place the lid on the cover to allow the atmosphere within the chamber to become saturated.
- With tweezers, place the TLC Plate into the chamber. Make sure that the Origin Line is above the solvent.
- Wait a few minutes. When you notice the solvent nearing the top of the TLC Plate (about a centimeter from the top), with tweezers, remove the plate from the developing chamber. Then mark the solvent front (the boundary between the dry and wet silica).
- With the tweezers, gently move the TLC Plate underneath a UV light source, chromatogram. You should be able to see dark spots. Outline the separated dots with a pencil to be able to locate the spots on the plate with visible light.
- Remove plate from the chromatogram and calculate the Rf value for each compound.
- Use a clean micropipette when switching to another solution to avoid cross-contamination between the lanes.
IV. Observations and Results
Lane SubstanceSpotted Distance movedby Spot 1 (mm)
Distance moved by Spot 2 (mm)
Distance moved by solvent (mm)
Rf value of Spot 1
Rf value of Spot 2 A Standard X 27 - 79 0 - B Standard Y - 59 80 - 0. C X + Y 28 59 79 0 0. D Unknown 28 - 80 0 - Table 2: Retention Factor Results (2) By calculating the Rf value of each lane, it is concluded that the Unknown substance had an Rf value that favors considerably with the Rf value of Standard X. So, the unknown substance s Standard X, meaning that the over the counter (OTC) analgesic is Tylenol.
more polar functional groups present will be less attracted to the less polar filtrate so more than likely it will be in the stationary phase, spending less time in the mobile phase which would then have a lower Rf vale. But this can also be related to the intermolecular forces, IMF, of the functional groups of the compound. A compound with a stronger IMF will spend more time in the stationary phase which would also equate to having a lower Rf value. 5
In the figures presented above, it is indicated that in Figure 1, acetylsalicylic acid (aspirin) is a polar molecule It is polar because of the functional groups present such as an ester functional groups which due to the two oxygens present contributes to the polarity. Another functional group present would be the carboxylic acid group which due to the two oxygens is polar and the OH leads to a polar O-H bond. The Intermolecular Forces of Attractions (IMF) present within Figure 1 would be Van der Waals, dipole-dipole and Hydrogen Bonding because of the C-OH bond present. Now, would this be water soluble? Organic molecules that are H soluble must contain at least one true hydrogen bonding site and contain fewer than six carbons. If a molecule contains one hydrogen bonding site but more than six carbons then the molecule will be water insoluble because there is a large polar region created by the carbon atoms. Acetylsalicylic acid does have one hydrogen bonding sites, but there are more than six carbons present. There are a total of nine carbons. Having more than six carbons allows there to be more of a non-polar region to be water insoluble. So, although there may be hydrogen bonding, this molecule would be water insoluble.
The same process can be done when we look at Figure 2, acetaminophen. Acetaminophen is a polar molecule. It is polar because of the oxygens and nitrogen present within the amide and alcohol functional groups. IMF present within would be Van der Waals, dipole-dipole bonding and hydrogen bonding because of the two hydrogen bonding sites present from the alcohol and amide functional groups. Note that when more hydrogen bonding sites are increased with an organic molecule, the more likely it is that it would water soluble even though there may be more than six carbons present. 6 In this case, acetaminophen would still be water insoluble, but it is more water soluble compared to the acetylsalicylic acid.
A concept to think about in the experiment is the concept of “like dissolves like.” Generally, “like dissolves like” explains that polar solvents will dissolve polar solutes while non- polar solvents will dissolve in non-polar solutes. 7 The two molecules above are both polar
substances that are likely to dissolve in the solvent, dichloromethane. The figure is presented below.
Figure 3 Dichloromethane (4)
Dichloromethane is the extraction solvent used in the experiment. It is a polar molecule because of the C-Cl and contains an IMF of Van der Waals and dipole-dipole. This allows both acetylsalicylic acid and acetaminophen to be polar soluble with the dichloromethane because they are all polar and do contain the same IMFs of Van der Waals and dipole-dipole.
Extraction, in general, is one of the most important techniques used a separation and purification process of organic compounds. It is when a solution is mixed with a second solvent that is usually insoluble with the first and done by mixing them together. 1 There is a solid liquid extraction within the first portion of the experiment in which Anacin, Excedrin and Tylenol were crushed up and extracted with dichloromethane. A solid-liquid extraction is the removal of a solute component from a solid by using a liquid solvent. 8 This allows the active ingredients, aspirin and acetaminophen to be within the dichloromethane. The solid portion of the extraction was either aspirin or acetaminophen while the liquid is represented by the dichloromethane. This allowed the aspirin and acetaminophen, the solids, to be pulled out of the drug since they were extracted with a polar solvent, dichloromethane due to the concept of “like dissolves like.” This would show which molecule was present in the unknown vile.
Thin Layer Chromatography, TLC, is a laboratory technique in which the mixture is dissolved within a solvent and placed near the bottom of a TLC plate. The TLC plate can be compromised of glass, metal or plates which is covered in a thin layer of adsorbent serving as the stationary phase. TLC also includes a mobile phase which a pure solvent or a mixture of solvents. The configuration of the mobile phase depends on the polarities of the compounds that are being separated. The stationary phase used the experiment is silica gel. It acts as a polar
Yet, they don’t move in the same way. This can be due to the stationary phase. There is a thin layer of silica gel coated with a thin layer of water. This results in silica atoms having -OH attached to them.
This has an extreme polarity and now also contains an IMF of hydrogen bonding and many hydrogen bonding sites. Organic molecules that are H20 soluble must contain at least 1 tru hydrogen bonding site and contain fewer than 6 carbons. Both molecules, aspirin and acetaminophen both contain hydrogen bonding and polar. Comparing them again, it is shown that there are two true hydrogen bonding on acetaminophen. Hydrogen sites can be induced with aspirin and its oxygen, but it only contains one true hydrogen bonding site. This concludes that the silica gel holding tightly onto acetaminophen relative to the aspirin. Aspirin will then more likely move with the ethyl acetate and is less tightly bound to the silica gel.
The final component to determine the identity of the unknown substance was calculated and visualized in Section IV. Observations. To identify distances, Rf (ratio of the front) formula was used. It is defined as the ratio of the distance traveled by the compound over the distance traveled by the developing solvent front as shown below. 1
Rf = distancetraveledbydevelopingsolvent frontdistancetraveledbycompound
The Rf value of a compound depends on its structure, but also to the nature of the adsorbent, silica gel, and solvent system, ethyl acetate. 9 Overall, compounds that have a low polarity have higher Rf values while high polarity compounds contain a low Rf value slowly move up the TLC plate while the non-polar components move up the plate at a faster rate. As shown in the observation section, Standard X has an Rf of 0 mm, Standard Y has an Rf of 0, X+Y together contain an Rf value of 0 mm and 0 and the Unknown Substance has an Rf of 0.
Solid Support
mm. By looking at the results, the unknown substance was most related to the distance of Standard X, in which this concludes that the OTC analgesic was Tylenol.
In conclusion, thin layer chromatography is a lab technique that can identify components that are in found in a mixture. On a TLC plate, there is a competition between the stationary phase, the silica gel, and the mobile phase, ethyl acetate. The more polar component will be held tightly by the silica gel and while other elutes up the TLC plate along with the ethyl acetate. This is dependent on polarity and the components IMF of the molecules being separated. Another component to think about is the structure of the molecule as organic molecules can be water soluble if they contain at least one hydrogen binding site and fewer than six carbons. This also goes with the concept with “like dissolve like.” By performing the experiment and calculating the Rf values, it can visualize which component makes up the unknown substance which can therefore conclude with OTC analgesic was identified.
8 Azad, Kalad. “About the Editor.” Advances in Eco-Fuels for a Sustainable Environment, 2019, p. xv., doi:10.1016/b978-0-08-102728-8-7.
9 Department of Chemistry. 2013, CHEM 334 Thin Layer Chromatography, www2.chem.wisc.edu/deptfiles/OrgLab/handouts/CHEM%20344%20TLC%20info.
Image Citations
Figure 1: Cacycle. “Acetylsalicylic Acid.” Wikipedia Commons, 2005, commons.wikimedia/ wiki/File:Acetyl_salicylic_acid_chemical_structure.
Figure 2: Wikipedia. “Paracetamol.” Wikipedia, 2017, en.wikipedia/wiki/Paracetamol.
Figure 3: OhnDoctor. “Dichloromethane.” Wikipedia, 2016, en.wikipedia/wiki/File:Dichloromethane_molecular_structure.
Figure 4: ChemSpider. “Ethyl Acetate.” ChemSpider, 2019, chemspider/Chemical- Structure.8525.
Thin Layer Chromatography Lab Report
Course: Organic Chemistry (3201)
University: Austin Community College District
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