Analysis of an Analgesic Tablet Using High Performance Liquid Chromatography (hplc)
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ANALYSIS OF AN ANALGESIC TABLET USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
Abstract
The purpose of this practical separate and calculate the constituents of an analgesic tablet anadin using reversed phase HPLC (C18 ODS column).
The main aim of the experiment is to quantify for the mass of aspirin and caffeine present in the anadin tablet sample using two different methods the internal standard calibration method and direct proportionality method. The value obtained for the internal standard method for caffeine present in anadin was 13.2mg and for the direct proportionality method was of 15.6mg. The value obtained for the internal standard method for aspirin present in anadin was 279mg and for the direct proportionality method was of 300mg. Comparing these to the value obtained from the commercial anadin tablet, 325mg of aspirin and 15mg caffeine, shows that internal method values calculated were closer to the values of the commercial box compared to the values achieved by direct proportionality method.
Introduction:
High-performance liquid chromatography (HPLC) is a widely used chromatography instrument which is also known as high pressure liquid chromatography. HPLC is used in analytical chemistry, biochemistry and forensic science.
High performance liquid chromatography is mainly an improved version of column chromatography. With HPLC, a pump of up to 400 atmospheres instead of gravity is required to allow the solvent to pass through the column, separating the mixture of compounds.
HPLC is based on the separation of molecules, which is achieved by the relative affinity of constituents of the mobile phase to the stationary phase.
The mobile phase is the solvent which flows over the fine silica beads that are coated with a thin layer of the stationary phase under high pressure. As the solvent travels through the column, they are separated at different degrees according to their interaction with the matrix silica material. The compounds with a high affinity with the stationary phase will interact more with the stationary phase and lag behind than those molecules with a low affinity and separation of the mixture is achieved. The compounds of the mixture travel at different rates due to their relative affinities with the solvent and stationary phase, and thus the sample injected will elute at separate bands (peaks). Moreover the separation improves, (resolution increases), as the length of the column increases. However the resolution may decline as the time taken for the solvent to pass through column increases, and therefore this increases the chance of results of diffusional spreading with the bands.
Outside the column is the detector, this is to detect the eluents, as they emerge from the column, they can be detected according to their UV absorption, refractive index or fluorescence. For this experiment ultraviolet detector was used, and thus from performing the HPLC a number of data were produced such as various peaks, retention times and area of peaks.
There are two main classes of HPLC depending on the relative polarity of the solvent and the stationary phase.
In the Normal phase HPLC, the stationary phase is made up of tiny polar compounds such as silica, and mobile phase is non-polar. Thus in this phase, the polar molecules in the solvent will stick to the polar stationary phase, and as a result the non- polar compounds will be eluted at early fractions. The principle of this Reverse phase is opposite to the Normal phase, as this method uses a non-polar stationary phase and polar mobile phase.
For this phase, the polar silica is modified to make it non-polar by chemically binding a hydrocarbon chain for example a common one being octadecylsilyl to its surface.
Thus in this type of HPLC, the elution order is reversed as the polar compounds are eluted first, as the non-polar compound remain held to the stationary phase.
The solution that is eluted from the column is constantly replaced the solution that is injected from the top of the reservoir.
For our practical the reversed phase HPLC (C18 ODS column) is used to separate and calculate the constituents of an analgesic tablet. This is further used to determine the quantitative components of aspirin and caffeine present in the anadin tablet using two calculations methods internal standard method and direct proportionality method.
Method
(refer to practical booklet)
Results
Figure 1: HPLC Chromatography for Unknown, Anadin
Figure 2: HPLC Chromatography for Aspirin
Figure 3: HPLC Chromatography for Caffeine
Calculations:
To show mass of Aspirin in Anadin tablet
1a) Direct Proportionality Method (not taking phenol into account)
Unknown: Anadin
Standard: Aspirin
300 mg of aspirin in 50ml of 40% MeOH
Concentration of aspirin:
300/50 = 6mg/ml
Half the concentration:
3mg/ml (added to ODS column for test)
10μl into machine, actual amount put in:
3/1000 x 10 = 0.03mg aspirin mass
Concentration present in 5μl of unknown:
(Unknown