Analysis of Analgesic Tablet by High Performance Liquid Chromatography
An unknown sample, 529, was tested using high performance liquid chromatography to detect the concentrations of acetaminophen, aspirin, and caffeine respectively. There was found to be 4.03±0.144mg/100mL of acetaminophen, 11.5±0.185mg/100mL of aspirin, and 4.89±0.185mg/100mL of caffeine. Based on accepted values, the maximum daily amounts of each compound are 4000mg of acetaminophen, 520mg of caffeine, and 400mg of aspirin. Introduction
This experiment was performed to determine the concentrations of acetaminophen, aspirin, and caffeine in the unknown sample 529. High performance liquid chromatography (HPLC) was the technique used. Using HPLC allowed for relatively quick runtime on each sample and highly accurate results. With HPLC, the mobile phase can be a part of dividing the components as opposed to other techniques such as gas chromatography where only the stationary phase is used to separate. Theory
The purpose of chromatography is the efficiently separate different components in a single sample. The difference in retention time between the two peaks divided by the average base width of the peaks, R, is used as a measure of separation between compounds. The minimum value of R is generally 1.0.
The factor K’ is also used for measurement. It can be considered as retention volume in terms of the volume of the component in the column. The higher the capacity factor K’ is, the higher the retention capacity of the column and in turn the longer the retention time for the component. Once the retention value (V₀) has been found it is divided into the net retention volume of the peak (V-V₀) to find k’ = (V-V₀)/V₀. Essentially, small k’ values are good for faster separation and larger k’ values have better resolution. From the view of maximum separation per unit time something between 2 and 6 would be a compromised k’ value. The longer a component stays in the stationary phase in the column, the better the separation will be however the peak will become broader and make the peak area less precise.
K’ is generally controlled in HPLC by altering the solvent strength in the mobile phase. Possibly even during the stationary phase. In gas chromatography, only the stationary phase is used for separation whereas in HPLC, the mobile phase can play part in that as well which can be an advantage towards using HPLC. If the k’ value is too small and the sample runs through too quickly after the first solvent, a list of relative solvent strengths must be checked and a weaker solvent chosen.
Polar stationary phases are normally used with non-polar solvents. With non-polar molecules like hydrocarbons or organic drugs, it is possible to get k’ values too low with every possible solvent. In that case it has to be changed to reverse phase HPLC which has a non-polar stationary phase and a polar solvent. That kind of increase in polarity needs a weaker solvent and therefore gives increased k’ values. To achieve optimum polarity for the separation of all components a mixed solvent composition can be used.
This experiment used reverse-phase HPLC and took place in an ion suppression mode. A 1% acetic acid buffer was used to suppress ionization of aspirin and also to increase the retention of acetaminophen and caffeine. Adjusting the ratio of methanol to buffer varies the mobile phase composition and can be done until proper separation of the components occurs. After being separated in the column, the components are individually detected by a UV absorption detector operated at 254 nm. Experimental
The unknown sample 529 was prepared before the lab from an analgesic tablet. One analgesic tablet was placed in an Erlenmeyer flask. A small amount of methanol was added and the tablet was crushed into small pieces with a stirring rod. It was filtered and the...
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