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C2507 Intensive General Chemistry – Spring 02 – E4: Aspirin Synthesis, HPLC Analysis
E4
Aspirin Synthesis
HPLC Analysis
E4 - Aspirin Synthesis. HPLC Analysis.
Salicylic acid was first discovered in the bark of willow tree. It was widely used to cure fever and headaches. Later it was demonstrated that salicylic acid and its derivatives (organic compounds called salicylates) affected the production of prostaglandins in the body. Excessive amounts of prostaglandins in the organism can cause inflammation, fever and pain. Salicylates are said to have antipyretic activity, lowering body temperature, but have little effect if body temperature is normal. More importantly, salicylates are mild analgesics, relieving headaches, neuralgia and rheumatism.
The use of salicylic acid was discontinued after the synthesis of aspirin. Its strong acidity had a series of side effects, mostly irritating the mucous membranes of the mouth and stomach. Although less acidic, acetylsalicylic acid can also cause the same problems to sensitive individuals, who use less acidic aspirin substitutes such as: acetaminophen and ibuprofen.
Common analgesics
In the composition of commercial analgesics tablets there is also caffeine and buffer excipients such as aluminum hydroxide Al(OH)3, and/or magnesium hydroxide Mg(OH)2.
Caffeine
This experiment has a dual purpose: first to introduce you to preparative organic chemistry and second to use HPLC (High Performance Liquid Chromatography), to test the purity of the obtained product. In the first part you will synthesize acetylsalicylic acid from salicylic acid and acetic anhydride. After purification of the final product you will also analyze some commercial analgesic tablets to discover the usefulness of this technique as a quality control tool.
Alcohols can combine with carboxylic acids under acidic conditions to form functional groups called esters. Esters can also be formed by the action of an organic anhydride (general formula RCO-O-COR) on an alcohol. One can easily make isoamyl acetate (better known as "Pear oil", used as a flavorant in mineral water and syrups) in this manner:
This reaction is very similar to that of isoamyl alcohol with acetic acid:
Both reactions are acid catalyzed, and both form isoamyl acetate. However, in the case of the acetic anhydride acetic acid is formed instead of water.
The reaction you will perform involves the exchange of a phenolic hydrogen for an acetyl (COCH3) group which is supplied by the acetic anhydride. The anhydride is split half form aspirin and the other half form acetic acid. The severe conditions (concentrated H2SO4 and heating) serve to speed the reaction. Following completion of the reaction, the aspirin must be freed from the solvent (water), acetic acid, and unreacted acetic anhydride. Because the solubility of aspirin decreases with temperature, purification can be accomplished by cooling the reaction mixture, to initiate the formation of crystals. Often, to promote crystallization, the sides and bottom of the flask are scratched to provide a microscopically rough surface where the molecules can adhere to and build up as crystals. The subsequent filtration of these crystals removes the acetic acid and any unreacted acetic anhydride.
Any remaining contaminants are removed by recrystallization in a solvent in which aspirin is sparingly soluble. Aspirin separates then as crystals leaving impurities behind in solution. This process should give a relatively clean product, whose purity can be determined by melting point analysis. A sharp melting point -when all the crystals melt over a 1 - 2 degrees temperature range signifies purity. The purity of the synthesized aspirin will also be tested by comparison to commercial aspirin using HPLC.
In this experiment, you will be introduced to HPLC through the analysis of analgesic tablets. First, you will chromatograph individual standard solutions of the tablets' components so that you can see their respective retention times. You will later compare the chromatograms of these standards and draw conclusions as of which one will be more retained based on its polarity. Then, you will choose a mixture of the standards and chromatograph that mixture under different conditions (e.g. different flow rate, detector attenuation, etc.) to observe the effects of these conditions on the chromatograms. Under the same conditions the aspirin sample you prepared should have the same retention time as the standard prepared with commercial samples.
High Performance Liquid Chromatography (HPLC)
Column chromatography, the oldest form of liquid chromatography, in which the stationary phase is packed in a column, and the eluting solvent allowed to percolate through the column under gravity, was refined greatly in the 1960's. During 1970's, most chemical separations were carried out using a variety of techniques including open-column chromatography, paper chromatography, and thin-layer chromatography. However, these chromatographic techniques were inadequate for quantification of compounds and resolution between similar compounds. During this time, pressure liquid chromatography began to be used to decrease flow-through time, thus reducing purification times of compounds being isolated by column chromatography. However, flow rates were inconsistent, and the question of whether it was better to have constant flow rate or constant pressure was debated; this lead to the development of high performance liquid chromatography, or HPLC. The technique is based on the same principles as column chromatography (i.e. partitioning between phases, the main difference is that the solvent is pumped through the column.
HPLC is further characterized by the following features:
In an HPLC system, a pump shoots the mobile phase through the system at the desired flow rate (Fig. 1).
Fig. 1. HPLC System Block Diagram
At the injector, the user inserts the sample for analysis. The injector is usually found right before the column. From there, the mobile phase picks up the sample and carries it through the column where it is separated into its various components. The separated components then travel through a detector cell where, based on the individual components' properties like UV absorption, fluorescence, or electrical conductivity, the amount of each component is determined. A recorder or another data-handling device translates the detector's electrical signals into peaks of various sizes.
The most popular mode of HPLC is reversed-phase chromatography (RP-HPLC). In this technique, the stationary phase is non-polar (e.g. carbon chain bonded to silica) and the mobile phase is polar (e.g. methanol, water, acetic acid). This is opposite to normal phase HPLC where the stationary phase is polar (e.g. silica, alumina) and the mobile phase is non-polar (e.g. hexane). The length of the carbon chain of the stationary phase used in RP-HPLC is typically eight (C8) or eighteen carbons (C18). The separation is based on partitioning of the sample components between the mobile and stationary phases.
Retention time, tR is widely used to identify the chromatographed peaks and the substances they represent. Under a given set of conditions, a particular compound will have a specific retention time. The unknown is compared to a known standard compound and, when the unknown is matched, the sample is identified. However, other compounds may have the same retention time. Confirmation can be obtained by varying the chromatographic conditions. Both the standard and the unknown should respond identically under the new conditions if they are, in fact, the same.
A similar technique involves co-injecting the unknown with a known standard. This is done by adding the standard component to the sample and injecting the mixture, the unknown component and the standard should show up as the same peak in the chromatogram if they are the same compound.
Other techniques used in advanced analytical methods include:
Once a peak is identified as an individual component, the next step would be to determine the amount of it present in the sample. This can be done from very accurate measurements of peak height or peak area. The concentration can then be determined using a calibration method. Peak height is measured from the baseline of the chromatogram to the peak maximum. The baseline is defined as the stabilized level of the recorder before the sample was introduced. The advantages of this technique are its simplicity and speed of calculation. However, there is a big disadvantage in that peak heights tend to vary much more than peak areas if chromatographic conditions vary. Peak areas also take into account any column degradation or longer retention, which results in wider, shorter peaks. Peak heights should only be used with symmetrical peaks. Peak areas may be determined by a variety of ways. The most widely used method being one-half the peak height times the peak width (i.e. the area of a triangle).
If all the components of a sample are detected, the concentration of each component in the sample may be expressed as a percent composition of the total sample:
The actual determination of the amount of a certain compound in a sample uses a calibration method. One such calibration method is called external standardization. An external standardization plot of peak height or peak area versus concentration is drawn based on chromatographing a series of standards of varying concentrations of the compound of interest under a given set of conditions.
Further information about HPLC can be found in Appendix A: Equipment and Techniques at the end of the manual and at the website. Also a tutorial on HPLC is available in the balance room of 302 Havemeyer.
Equipment and Reagents
Salicylic Acid Acetic Anhydride
Concentrated H2SO4 10mL vials
125 mL filtration flask, Hirsh funnel, 1.3 cm filter paper and #3 neoprene adapter
2mL pipettes 10�L micro syringe (blunt-pointed) Ibuprofen Acetaminophen
Acetylsalicylic Acid Caffeine
Bayer� aspirin Anacin�
Excedrin� HPLC System
Mobile Phase: 0.4% triethylammonium acetate, 13.8% methanol, and 85.8% H20
Part I-a— Synthesis of Aspirin
Safety Note: Avoid contact with acetic anhydride and sulfuric acid. They cause severe burns and destroy cloth. Acetic Anhydride is very hygroscopic, close the flask immediately after use.
Part I-b—Isolation and Recrystallization of Aspirin
Part II —HPLC Analysis
HPLC analysis will be simpler after using the HPLC tutorial PHENOMENEX.
Basic Chromatographic Conditions:
Column: | Pecosphere 3x3 C18 |
Detector: | UV (254 nm) |
Mobile Phase: | 0.4% triethylammonium acetate, 13.8% methanol, and 85.8% water. |
Exploring some HPLC Parameters
For this part of the experiment you will work exclusively with the 1:4:10 mixture prepared in 7. Keep your printouts for analysis.
Bring the flow rate back to its original 2.00 mL/min and wait a few minutes for the system to equilibrate. Switch your chart speed to 30 mm/min and re-inject the sample. Try it again with the chart speed at 10 mm/min. Be sure that the switch on the recorder for adjusting the chart speed is locked into the desired speed. If the switch is at an "in between" position, the recorder will not work properly.
Finally, analyze your sample at an attenuation value (AUFS, Absorbance Units at Full Scale) higher and lower than the sensitivity used at the beginning of the procedure.
Notes:
TABLET | COMPONENTS |
Anacin | Aspirin; caffeine |
Tylenol | Acetaminophen |
Bufferin | Aspirin |
Nuprin | Ibuprofen |
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