High Performance Liquid Chromatography Analysis of Aspirin
Was aspirin (acetylsalicylic acid) successfully synthesized? Are there impurities or by-products present in the synthesized aspirin? How pure is the synthesized aspirin?
In the last experiment, aspirin was synthesized followed by characterization of the product using several different techniques. Melting point was a test that provided information about the identity and purity of the aspirin product. The iron(III)chloride test was used to detect the presence of a phenol group, with a positive result indicating the reactant salicylic acid was leftover as an impurity. Thin layer chromatography (TLC) was also used to determine if the white powder synthesized was aspirin and if there were any impurities or by-products in the final sample. In this experiment, high performance liquid chromatography (HPLC) will be used to further characterize the aspirin product. It is similar to TLC in that it has the ability to separate components of a mixture and identify them. However, HPLC offers several advantages over TLC. HPLC has greater separation capabilities, lower detection limits, and can be used to quantify the amount of substance present. It also can be fully automated.
Liquid chromatography has become a widely used analytical technique because of its ability to produce high resolution separations of non-volatile compounds. High performance liquid chromatography (HPLC) uses a high pressure to force solvent through packed columns containing fine particles coated with stationary phase. The analyte molecules are separated as they flow down the column based on differences in interactions with the stationary phase. The HPLC column is typically 5-30 cm in length and 1-5 mm in inner diameter. The interior of the column is packed with spherical, microporous particles with diameters ranging in size from 3-10 μm. The average human hair is about 70µm in diameter, thus approximately 10 stationary phase silica particles could line up across a human hair. Reversed-phase liquid chromatography, the most common technique for separating organic compounds, utilizes a non-polar stationary phase and a polar solvent which often gradients to a more non-polar solvent during the separation. A common stationary phase is octadecyl (C18), which is covalently attached to the surface of the silica stationary phase particles. Figure 1 shows a representation of a typical HPLC column and stationary phase.
Figure 1: A typical HPLC column and stationary phase particle are shown.
The HPLC instrument consists of solvent reservoirs, solvent pumps, a sample injection valve, a high pressure column, a detector, and a computer to control the system and display the results. (Figure 2). There are typically 3-4 solvent reservoirs for an HPLC system which contains solvents of varying degrees of polarity and viscosity. Very pure HPLC-grade solvents are required to minimize background signals from impurities. The HPLC pump can perform an isocratic elution in which the various solvents are mixed in a fixed percentage during the chromatographic run or the pump can also be programmed to perform a gradient elution in which the solvents are mixed in different proportions during the course of the chromatographic run. Quality HPLC pumps produce a steady flow rate, thus reducing detector noise which can swamp weak analyte signals. A typical pump contains two pistons that produce a programmable, constant flow rate up to 10 mL/min and pressures up to 400 bar. An injection valve can be used to deliver precise volumes of sample in the range 2-500 uL using different sized injection loops. Many types of detectors can be used in HPLC analyses and the detector in the C103 laboratory is based upon absorbance of UV or Visible light.
Figure 2: Photograph of a HPLC system.
The output from an HPLC is called a chromatogram, which is a plot of detector response versus time. Figure 3 shows an...
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