DETERMINATION OF ASPIRIN IN ANALGESIC TABLET BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Razel Elaine Grace A. Catalua Sarah Jane V.

Veloso ABSTRACT A reverse phase high performance liquid chromatography method was developed for the analysis of aspirin in analgesic tablet. The purpose of this experiment was to discuss the basic principles and concepts involved in the use of HPLC and to identify the peak of aspirin in the chromatogram. The separation was achieved by octadecyl column (C 18) and acetonitrile/water (1:9 v/v) as eluent, at a flow rate of 1.5 mL/min. Detection was carried out at 5-um particle size and ultraviolet detection at 254 nm. The significant peaks as shown in the resulting chromatogram in the experiment with 1.433RT and 10.342RT are the most probable peaks of aspirin based on literature. The method has been validated that the acetonitrile solvent is probably not effective to the determination of the aspirin component in analgesic tablet for it shows no sharp peaks at all. The accuracy of the method depends on the significance of the solvent that has greatly to do with the affinity of the sample to be determined. INTRODUCTION High performance liquid chromatography is basically a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster. It also allows the use of a very much smaller particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the components of the mixture. The other major improvement over column chromatography concerns the detection methods which can be used. These methods are highly automated and extremely sensitive. There

surface - typically with either 8 or 18

carbon atoms in them. A polar solvent is used - for example, a mixture of water and acetonitrile. In this case, there will be a strong attraction between the polar solvent and polar molecules in the mixture being passed through the column. There would not be as much attraction between the hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in the solution. Polar molecules in the mixture will therefore spend most of their time moving with the solvent. Reversed phase HPLC is the most commonly used form of HPLC1. Looking at the whole process of the flow scheme for HPLC.

are two variants in use in HPLC depending on the relative polarity of the solvent and the stationary phase. In reverse phase, the column size is the same with the normal phase, but the silica is modified to make it non-polar by attaching long hydrocarbon chains to its

Figure 1. Flow Scheme for HPLC1

HPLC utilizes different types of stationary phase typically of the hydrophobic saturated carbon chains. A pump that moves the mobile phase(s) and analyte through the column, and a detector that provides a characteristic retention time for the analyte. The detector may also provide other characteristic information (i.e. UV/Vis spectroscopic data for analyte). Analyte retention time varies depending on the strength of its interactions with the stationary phase, the ratio/composition of solvent(s) used, and the flow rate of the mobile phase2. In this experiment, High Performance Liquid Chromatography will be used to determine the composition of aspirin in analgesic tablet. Chromatography is a technique used to separate and identify individual components in a mixture. Compounds stick to reverse phase HPLC columns in high aqueous mobile phase and are eluted from reverse phase HPLC columns with high organic mobile phase. Many HPLC methods use acetonitrile as part of the mobile phase. It is an excellent eluent. It has low viscosity, good selectivity, 100% miscibility with water, reasonable buffer solubility, and is almost transparent to UV light.

fully fill a 10-mL loop on the injector port. Analyses are carried out on a C-18 silica column, 25 cm x 4.4 mm (5-um particle size) Supelco Analytical No. 018955 AE with guard column. Perkin Elmer UV/VIS Spectrophotometric Detector LC290 is set at 254 nm to monitor absorbance of the effluent. The integrator is operated at a running time speed of 5 minutes to record chromatograms and peak areas. Reagent Preparation Fifty-five mL of HPLC-grade acetonitrile was diluted to volumetric flask with HPLC-grade water having a ratio of 1:9. Prepared sample was then placed in a capped amber bottle. Sample Preparation Analgesic tablet was ground into a fine powder with a clean mortar and pestle. Weighed approximately 0.2502 g and dissolved in 25 mL of HPLC solvent with gentle heating. Cooled to room temperature and diluted to volume with HPLC solvent. Five mL of the solution was diluted to 50 mL with HPLC solvent in a volumetric flask. Qualitative Analysis A chromatogram of 10 uL of the analgesic tablet solution was recorded. Ten uL analgesic solution was injected into HPLC. Observed peaks grow and finally identify which peak is the aspirin. RESULTS & DISCUSSION During the course of elution, the chromatogram showed five peaks that were integrated by the system (Figure I). The retention time of the first peak was 1.433, followed by 4.148, 6.125, 10.341, and 14.155. This was through a 1:9 v/v acetonitrile/water. Other chromatographic conditions include analyses carried out on a C-18 silica column, 25 cm x 4.4 mm (5-um particle size) Supelco Analytical No. 018955

MATERIALS AND METHODS Reagents In performing the determination of aspirin in analgesic tablet by high performance liquid chromatography, acetonitrile HPLC-grade solvent and HPLCgrade water was produced in our laboratory. Chromatography Conditions Operating

Perkin Elmer Binary LC Pump 250 is used to deliver the eluent at a constant flow rate of 1.5 mL/min. Solutions are injected to

AE with guard column. Perkin Elmer UV/VIS Spectrophotometric Detector LC290 is set at 254 nm to monitor absorbance of the effluent. The integrator is operated at a running time speed of 5 minutes to record chromatograms and peak areas. Various solvent systems can be used to analyze aspirin content in a sample. In some literature, Acetonitrile/methanol:20 mM phosphate buffer at pH 3 (50:7:43 v/v) as eluent, aspirin was eluted in 9.27 min2. In a diode array detector with 4-21% acetonitrile as solvent, aspirin was eluted in 4.00 minutes3. In principle, liquid chromatography and HPLC work the same way except the speed, efficiency, sensitivity and ease of the HPLC is highly superior. The solvent system plays an important role in chromatography. In order for good separation to happen, the solvent system should me miscible with each other like acetonitrile in water or methanol in water. Mixing an immiscible water/toluene would create a mess in the column. This solvent system operates in like dissolves like principle where polar compounds dissolve other polar compounds and nonpolar to nonpolar compounds. The solvent system should be able to dissolve the component, which needs to be separated. As elution happens, components in the sample separate according to its affinity with the solvent. There should be significant difference of the components affinity towards the stationary phase and mobile phase of the system. In a Reverse Phase Chromatography where the stationary phase is nonpolar with a relatively aqueous or moderately polar mobile phase, the least polar tend to be eluted longer than other components4. The relatively polar solvent gets eluted with the most polar component, which is dissolved in it. Moreover, the less polar the component in a sample, the longer it gets eluted. In a chromatogram, the longer retention time tells us that the component

has less affinity towards the solvent and is less polar in a Reverse Phase Chromatography). Aside from mobile phase surface tension (organizational strength in eluent structure), other mobile phase modifiers can affect analyte retention. For example, the addition of inorganic salts causes a moderate linear increase in the surface tension of aqueous solutions (ca. 1.5 107 J/cm per Mol for NaCl, 2.5 107 J/cm per Mol for (NH4)2SO4), and because the entropy of the analyte-solvent interface is controlled by surface tension, the addition of salts tend to increase the retention time. This technique is used for mild separation and recovery of proteins and protection of their biological activity in protein analysis (hydrophobic interaction chromatography, HIC) 5. Structural properties of the analyte molecule also play an important role in its retention characteristics. In general, an analyte with a larger hydrophobic surface area (C-H, C-C, and generally non-polar atomic bonds, such as S-S and others) results in a longer retention time because it increases the molecule's non-polar surface area, which is non-interacting with the water structure5. Retention time increases with hydrophobic (non-polar) surface area. Branched chain compounds elute more rapidly than their corresponding linear isomers because the overall surface area is decreased. Similarly organic compounds with single C-C-bonds elute later than those with a C=C or C-C-triple bond, as the double or triple bond is shorter than a single C-C-bond5. Another important component is the influence of the pH since this can change the hydrophobicity of the analyte. For this reason most methods use a buffering agent, such as sodium phosphate, to control the pH. The buffers serve multiple purposes: they control pH, neutralize the charge on

any residual exposed silica on the stationary phase and act as ion pairing agents to neutralize charge on the analyte. Ammonium formate is commonly added in mass spectrometry to improve detection of certain analytes by the formation of ammonium adducts. A volatile organic acid such as acetic acid, or most commonly formic acid, is often added to the mobile phase if mass spectrometry is used to analyze the column eluent. However, a buffer system was not used in the experiment. The effects of acids and buffers vary by application but generally improve the chromatography6. In the literature in which the experiment was based, a solvent system of water:acetonitrile:triethylamine:glacial acetic acid in 94.1:5.5:0.2:0.2 (v/v/v/v) was used, the results yielded a retention of 1.4 minutes for aspirin. The retention time of 1.433 minutes and 10.342 minutes are the most reasonable peaks integrated in the chromatogram. CONCLUSION With the results shown, there is significant influence of proper solvent system to be used in high performance liquid chromatography. There was no standard calibration done before running the sample making other established experiments as the reference of the retention time of aspirin. The significant peaks as shown in the resulting chromatogram in the experiment with 1.433RT and 10.342RT are the most probable peaks of aspirin based on literature. REFERENCE 1 http://www.chemguide.co.uk/analysis/chro matography/hplc.html. July 27, 2010. 2 http://en.wikipedia.org/wiki/Highperformance_liquid_chromatography. July 27, 2010. 3 http://www.ijpsonline.com/article.asp? issn=0250474X;year=2007;volume=69;issu

e=4;spage=597;epage=599;aulast=Ananda kumar, July 27, 2010 4 http://jpet.aspetjournals.org/content/319/3/1 467.full, July 28, 2010 5 http://en.wikipedia.org/wiki/Highperformance_liquid_chromatography, July 28, 2010 6 http://en.wikipedia.org/wiki/Highperformance_liquid_chromatography#Rever sed-phase_chromatography_.28RPC.29, July 15, 2010