1. Synthesis and Analysis of Aspirin
In the second part of this experiment we will determine the purity, and confirm the identity, of the aspirin product from last week.
Chemistry 1060 Laboratory

2. Analysis Purity determination Thin Layer Chromatography (TLC)
Separation and identification
stationary phase – silica gel
mobile phase – organic solvent
Relative attraction to the two phases
Polarity
Much like the paper chromatography we did last semester, thin layer chromatography, or TLC, is a chromatographic method for separating and identifying the components of a mixture according to their chemical structure. In TLC, the stationary phase is commonly silica gel, and the mobile phase is an organic solvent.
As the mobile phase moves up the silica gel plate, compounds will travel at different rates depending on their relative attraction to the two phases.
The polarities of the compounds, the solvent, and the stationary phase will all contribute to the rate at which compounds travel up the TLC plate.

3. Analysis Thin Layer Chromatography (TLC) Retention factor (Rf) values
3 cm
5 cm
Solvent front
Rf = 3/5 = 0.60
Origin
The Retention factor, or Rf value, will be calculated for the compounds once the TLC is developed.
These values are used for comparison with the known compounds involved in the reaction.
The origin, or baseline, is where the samples are spotted on the TLC plate, and the solvent front is the distance the solvent has traveled during development of the plate.
To calculate the Rf value, mark the center of the spot, and divide the distance from the origin to that spot, by the distance from the origin to the solvent front. In this example, the spot has traveled 3 cm from the origin, and the solvent front has traveled 5 cm, so the Rf value is .6.

4. Analysis UV Thin Layer Chromatography (TLC)
Salicylic acid (starting material)
Pure aspirin
Isolated product
UV detection
Results
Rf of pure aspirin
salicylic acid removed? UV
In this experiment, we will spot our TLC plate with three substances for comparison:
The starting material, salicylic acid,
the known product, aspirin, which will be provided by the stockroom
and the product we isolated last week.
Since both the starting material and the product are colorless, that is, they do not absorb in the visible region of the spectrum, we will not be able to detect them with room light. Instead, we will use ultraviolet absorption to detect the spots. TLC plates are impregnated with a fluorescent material, and glow green under UV light. Since the organic compounds in this experiment do absorb in the UV, they will show up as dark spots. While viewing the TLC plate under the UV lamp, you should circle the spots with a pencil in order to determine their Rf values later.
If our isolation and purification have gone well, we should observe the presence of aspirin in our product, but we should not observe the presence of salicylic acid. The Rf values for these compounds will be calculated and reported in the post-lab.

5. Analysis Structure confirmation
1H nuclear magnetic resonance (1H NMR) - or proton magnetic resonance (PMR)
To further confirm the identity of our product, we will use a spectroscopic technique call nuclear magnetic resonance. NMR is based on the different energies of nuclear spin states in a magnetic field, and while there are several nuclei with spins that can be analyzed by this technique, we will focus on proton magnetic resonance.
When protons are placed in a magnetic field, their spins can align either with or against the field. These are called the alpha and beta spin states, respectively. Aligning with the field represents a lower energy state, and against it a higher energy state

6. Analysis
1H NMR
In the absence of a magnetic field, the spins of all the protons in a sample are oriented in random directions. When an external magnetic field is applied and they align with or against it, the difference in energy between the alpha and beta states increases as the magnetic field strength increases. The magnetic field used in modern NMR spectrometers may be as much as 250,000 times stronger than the Earth’s magnetic field.

7. radio-frequency region of the spectrum
Analysis
1H NMR
radio-frequency region of the spectrum
A proton may be excited from a lower energy state to a higher one by absorption of light at the frequency corresponding to the difference in energy between the two states, as determined by Planck’s equation.
This frequency of light is in the radio frequency region of the electromagnetic spectrum.
To measure an NMR spectrum, the sample is placed between the poles of a strong magnet, and irradiated with radio frequency light. Those frequencies that are absorbed by the sample are detected and plotted on a printer.

8. Analysis 1H NMR – Shielding
A naked proton will experience the full external magnetic field, but of course protons are surrounded by electron clouds. An electron cloud in the magnetic field will circulate and create an internal, induced magnetic field that opposes the external magnetic field, and therefore shields the proton from the external magnetic field. In order for the proton to absorb at the same frequency, we will need to increase the strength of external magnetic field.
The density of the electron cloud surrounding a proton will be affected by electron-withdrawing atoms. In methanol, for example, the electronegative oxygen withdraws electron density more strongly from the proton attached to it, than from the further away protons attached to the carbon. We say that these protons are in different chemical environments. The hydroxyl proton will be less shielded by electrons and will absorb at a lower field strength, while the methyl protons are more shielded and absorb at a higher field.

9. Analysis 1H NMR – Chemical Shifts, Chemical Equivalency
tetramethylsilane
(TMS)
The NMR spectrum is plotted with increasing magnetic field strength toward the right, so we refer to the signal from the more shielded methyl protons as being upfield, and that from the less shielded hydroxyl proton as being downfield. The position in the spectrum is called the chemical shift. Note that the three methyl protons give rise to only one signal, since they are all in the same chemical environment. We refer to them as being chemically equivalent.
The small signal at the far right is due to an internal standard, tetramethylsilane, or TMS, that is added to the sample for the purpose of calibrating the NMR instrument.

10. Analysis 1H NMR – Chemical Shifts, Integration 3 1
Without going into detail on the units, the NMR scale is measured in parts per million, downfield from TMS. This is called the delta scale. It usually ranges from zero to ten parts per million, but sometimes may be recorded up to 12 parts per million. The chemical shifts of the protons in methanol are at about 3.4 and 4.8 parts per million. Note also that the three methyl protons give rise to a more intense signal than the one hydroxyl proton.
In fact, the integrated areas under these peaks will be in a 3 to 1 ratio, proportional to the number of chemically equivalent protons in the compound. The integrated areas of the peaks are often printed on the NMR spectrum.

11. Some typical values of chemical shifts are shown in this table
Some typical values of chemical shifts are shown in this table. There is also a chart in your lab manual. Note that these values are approximate, and may be strongly affected by neighboring deshielding groups. Acidic protons, such as those on carboxylic acids, alcohols, and phenols, may be quite variable.
In our methanol example, we saw that the protons attached to the carbon were at 3.4 ppm, which is common with either halogen atoms or oxygen atoms attached to that carbon,
and that the hydroxyl proton was at 4.8, in the range of alcohol protons.

12. Analysis 1H NMR – Chemical Shifts, Complex Patterns
Aromatic protons, or protons attached to a benzene ring, have chemical shifts between about 7 and 8 parts per million. Note that in this spectrum of toluene, the three methyl protons appear as a single peak, since they are chemically equivalent, but the five aromatic protons give rise to a complex pattern of peaks. This is due to a phenomenon called splitting, but we will not go further into that in this course. We should simply note that aromatic protons can give rise to complex patterns of peaks.

13. Analysis 1H NMR – Characterizing starting material and product
salicylic acid
aspirin
In this experiment, we will plot NMR spectra for both the starting material, salicylic acid, and the product, aspirin, and then identify which protons in these two compounds give rise to the signals in the NMR spectra. Note that the aromatic protons, show in blue, and the carboxylic acid protons, in green, should look fairly similar in both spectra. The larger difference in the spectra will be due to the phenolic proton in the starting material, and the methyl protons in the product, shown in red.

14. Summary Week 2: Goals Week 1:
To synthesize aspirin, from salicylic acid and acetic anhydride
To purify the product using recrystallization
Week 2:
To verify the purity and identity of the product using thin layer chromatography (TLC)
To further confirm the identity of the product by nuclear magnetic resonance (NMR) spectroscopy
In summary, in the first week of this experiment we synthesized and purified aspirin,
and in the second week, we will verify the purity and identity of the product using thin layer chromatography,
and further confirm the identity of the product using nuclear magnetic resonance spectroscopy.
Good luck, and have fun!