(CHE 276) Organic Chemistry Lab Experiment #7

Unknown Identification and Characterization of Self-assembly

— Dr. Luk

In this lab, students will use thin layer chromatography and knowledge of ranking the polarity of organic molecules to identify unknown compounds. Students will also use polarizing microscope to characterize the self-assembly of surfactants and organic salts in water – prepared last week.

Identification of Unknown compounds

Here, students will be given a solution containing three known molecules, benzophenone, 2,4,6-tribromophenol, and 4-methybenzyl bromide, structures of which are shown below. This solution will serve as a reference for determining the unknown compounds.

A solution containing two of the following three molecules will also be given to the students. By using TLC and comparing to the reference molecules (see above), the student will determine which two of three molecules are present in his or her unknown solution.

Students will be given three eluent solvents with different polarity, and must use the reference compounds to determine which solvent is the best for their separation and thus for identifying the unknowns. To achieve these two goals, students will first, just by examining the structures, determine relative polarity of the three reference molecules, and of the three molecules in the unknown solution. Then, by using thin layer chromatography, and comparing

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the relative position of the reference and unknown sample, the student will use simple logic to determine the identity of the two molecules in the unknown sample.

How to compare and determine relative polarity between two different organic molecules?

The polarity of a small organic molecule is determined by the functional groups the molecule possesses. The symmetry of the molecule also plays a small role in influencing the polarity of the molecule. Below is a list of rough general guidelines for ranking the polarity from high to low.

1. Charged groups such as R4N+ or COO- are the most polar functional groups. 2. Functional groups with hydrogen bonding capability are the second category of polar

functional groups. 3. Atoms in functional groups with high electronegativity introduce more polarized bonds

(and thus more polar molecules) than those with low electronegativity. 4. The more polar functional groups a molecule possess, the more polar the molecule will

be. 5. In general, a polar functional group has a more dominating effect over less polar

functional groups on a molecule. For example, a carboxylate (RCOO-) is likely more

polar than a sugar-based surfactant that has five hydroxyl (OH) groups. 6. All else being the same, an asymmetric molecule is more polar than a symmetric

molecule.

For your reference, a periodic table of electronegativity is included below.

Image extracted from http://en.wikipedia.org/wiki/Electronegativity

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Procedures:

1. Obtain and transfer roughly 1 mL (about a small squeeze of a pipet bulb) of the reference solution and 1 mL of the solution of an unknown sample in two different vials. You will be assigned to a specific unknown sample.

2. Run the TLC of the reference compounds three times, each time with a different eluent solvent: 100% hexane, 1:20 ethyl acetate: hexane, and 1:20 CH3OH:CH2Cl2.

3. Run the TLC of the unknown and the reference compounds on the same plate using the best solvent you determined from step 2.

4. Draw and record the results from step 3 in your notebook.

Characterization of Molecular Self-assembly by Using Polarizing Microscopy.

For most assemblies of molecules, such as a snow flake, molecular crystals, liquid crystals, micelles, mammalian cell membranes, etc, the molecules are ordered in some way, rather than randomly oriented in a sample. Likewise, if the composition of the sample prepared last week is right, assembly of molecules will occur, and the molecules (specifically 5ꞌDSCG and the surfactants) will have certain orientation order as shown in the manual for the previous Lab. To determine if such molecular or atomic order exists in a sample, many scientific techniques have developed. Among these techniques, polarizing light microscopy is commonly used and powerful for characterizing the order in the sample.

Polarizing Light Microscopy

Light is an electromagnetic wave. Normal light has waves oscillating in all directions perpendicular to the direction of propagation of light. Waves oscillating in some directions can be blocked (or filtered) more than other directions by highly ordered material, such as a thin film of crystals, leaving light waves oscillating only in a certain plane. The thin film of crystals is called a polarizer (or polaroid), and the plane of light is call plane polarized light (Figure 1).

Figure 1. Nonpolarized light passing through a polarizer, becoming plane polarized light. When plane polarized light passes through a second polarizer with the materials aligned perpendicular

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to the first polarizer, all lights are blocked out. Image extracted from: http://www.one-school.net/Malaysia/UniversityandCollege/SPM/revisioncard/physics/wave/electromagnetic.html

This phenomenon is due to the light being absorbed more by one axis of the ordered materials than the other axes. A good polarizer contains materials that are so ordered that, except for one plane of oscillation, light waves oscillating in all other planes are absorbed. Thus, when two such polarizers are stacked together with the alignment of the materials at 90o relative to each other (crossed polarizers), all lights are blocked out, and the stacked polarizers appear to be black. Alternatively, when one of the polarizers is rotated away from 90o, light starts passing through again, and one can see through the polarizers (Figure 2).

Figure 2. Light can pass through polarizers that are held in parallel to each other (A) and light is blocked when polarizers are perpendicular to each other (B). Any sample containing ordered molecules put in between the crossed polarizers may cause the light to pass through again. Image extracted from: http://micro.magnet.fsu.edu/optics/intelplay/qx3polarization.html

When ordered materials, such as liquid crystals or crystals, are placed in between the polarizers, the plane polarized light passing through the first polarizer will be rotated again by the liquid crystals to another angle, and thus will pass through the second polarizer. Note that, when the long axis of the molecular order in the sample aligns parallel to the axis of either one of a pair of crossed polarizers, the plane polarized light will not be rotated, and no light will pass through. In such a case, one just needs to rotate the sample a little bit between the polarizers, which will once again allow plane polarized light to pass through.

A polarizing light microscope consists of two polarizers. One is located above, and the one below, a sample stage that can be rotated. One of the polarized can be rotated to create crossed polarizers and parallel polarizers, and any angle in between.

Materials that contain no order in their molecular or atomic composition, such as liquid water or a piece of glass, will have the same property no matter which direction a measurement is made on the sample. Such material is said to be isotropic. For example, glass or liquid water has only one refractive index. Between cross polarizers, these materials will not change the direction of plane polarized lights, and thus the sample will appear black. Other materials that

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have ordered molecular or atomic compositions, such as calcite (calcium carbonate) or a liquid crystal will have different results when one measures the materials from different directions. These materials are called anisotropic. For example, when the refractive index is measured for a liquid crystal, two different (and extreme) values will be obtained, one is from measurement carried out parallel to the alignment axis, and the other is from measurement perpendicular to the alignment axis. Materials that have two refractive indexes are also called birefringent. When positioned between a pair of crossed polarizers, these materials will show a light pattern that is governed by the molecular or atomic organization order in the sample. For liquid crystals, the organization of the molecules in the sample can be elucidated by the images (optical micrographs) by using a polarizing microscope, often with crossed polarizers.

In this lab., we will study whether a special kind of liquid crystal will or will not form depending on whether the composition is right for the assembly of different molecules to form in water.1,2 The composition was pre-determined and the sample was prepared in the previous lab. Only samples that show no or little precipitate in the vial should be taken for observation under a polarizing microscope. All isotropic samples will appear dark, and stay dark when the sample is rotated on the stage. Anisotropic samples (i.e. self-assembly of molecules occured) will allow some light to pass through, and show crossed or ellipsoidal images, and sometimes, may be colored.

Procedures for preparing optical cells of surfactant and 5ꞌDSCG in water, and use of the polarizing microscope.

Here, you will work with a partner for preparing and using the same optical cell, you will still have your own result and observation for the report.

1. Cut a piece of Saran wrap about the size of the glass slide. Using a hole-puncher, punch four holes in Saran wrap. Put the Saran wrap on top of one of the glass slides to create four wells. Two of the four wells will be for each of the two partnered students. One well is for the control sample, and the other one is for the test sample.

2. By using a glass rod or pipette, each student must transfer a small amount of his control and test sample into the two separate wells. The other student does the same for his two wells. Put another glass slide on top of samples (two controls and two tests) so the sample and the Saran wrap are sandwiched in between glass slides. Remember to label the position of the wells on your notebook, so the wells can be identified easily.  

3. Apply six binder clips on the four edges of the glass slides, and remove one wire of each binder clip only on one side of the glass assembly. 

4. Make observation using a polarizing microscope, write down the observation in the table you made in the previous lab.  Be sure that the sample is placed under crossed polarizers – when viewing through the eyepiece, the air (no sample) should appear dark (example, see Figure 2).

5. Disassemble the binder clips/glass slides assembly.  Return the binder clips to a box, and discard the glass slides, Saran wrap and the samples to the proper disposal container (glass waste container).

References

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(1) Simon, K. A.; Sejwal, P.; Gerecht, R. B.; Luk, Y.-Y. Water-in-Water Emulsions Stabilized by Non-Amphiphilic Interactions: Polymer-Dispersed Lyotropic Liquid Crystals Langmuir 2007, 23, 1453-1458.

(2) Varghese, N.; Shetye, G. S.; Bandyopadhyay, D.; Gobalasingham, N.; Seo, J.; Wang, J.-H.; Theiler, B.; Luk, Y.-Y. Emulsion of Aqueous-Based Nonspherical Droplets in Aqueous Solutions by Single-Chain Surfactants: Templated Assembly by Nonamphiphilic Lyotropic Liquid Crystals in Water Langmuir 2012, 28, 10797-10807.

http://www.microscopyu.com/articles/polarized/index.html

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