Determination of Aspirin Using Two Chromatographic Method Using (Hplc or Gc) And Two Spectrophotometric Method.

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Last Updated: 13 Feb 2023
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Determination of aspirin using two chromatographic method using (HPLC or GC) and two spectrophotometric method.


Aspirin, the most widely used drug in the world, has been known to mankind for over a century. It is not only the pharmacologically active entity, but is also bio transformed into a major metabolite, i.e. salicylic acid, which also exhibits similar.

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Pharmacological properties. Hence it is necessary to quantitate aspirin along with its metabolite(s) in various biological matrices accurately and precisely to correlate with pharmacological activity This paper provides a comprehensive overview of various bioanalytical methods (HPLC, GC and spectrophotometric methods) that have been reported for direct quantitation of aspirin along with its metabolite(s). the HPLC That developed method is rapid and sensitive and therefore suitable for determination of analgesic drugs and other drugs. The instrument used for carrying out HPLC was Shimadzu LCVP2010C integrated system equipped with quaternary gradient pump, Column utilized Kromasil C18 (180 X4.6mm),5µm,and the detection made possible using UV-Vis detector at a wavelength of 277nm and the mobile phase employed was acetonitrile: methanol 60:40(v/v).The Rt(Retention time) of aspirin was found to be 4.303. (1)

Spectrophotometry is the process by which radiation from the ultraviolet and visible regions of the electromagnetic spectrum are utilized to quantitatively measure. and one of spectrophotometric method sensitive, fast and simple for the determination of aspirin is proposed. The method was based on oxidation-reduction reaction of aspirin with potassium permanganate with maximum absorption at 350 nm.


[image: ]The common pain reliever Aspirin is known chemically as acetylsalicylic acid and has been in use as a pharmaceutical agent for over 100 years (2). Aspirin is used to relieve mild to moderate pain, reduce fever, to reduce inflammation and swelling in conditions such as arthritis Aspirin was used in low doses as a blood thinner to prevent the formation of blood clots. It is effective in reducing the risk of stroke and offers a protective effect against heart attacks in men with chest pain. It is unique among Cox-inhibitors (cyclooxygenase) because it covalently modifies the proton of enzymes and irreversibly inhibits them.


Chromatographic process : Chromatographic process can be defined as separation technique involving mass-transfer between stationary and mobile phase. HPLC utilizes a liquid mobile phase to separate the components of a mixture. The stationary phase can be a liquid or a solid phase. These components are first dissolved in a solvent, and then forced to flow through a chromatographic column under high pressure. In the column, the mixture separates into its components. The amount of resolution is important, and is dependent upon the extent of interaction between the solute components and the stationary phase. The stationary phase is defined as the immobile packing material in the column. The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide [image: ]variety of chemical mixtures HPLC is a dynamic adsorption process. Analyte molecules, while moving through the porous packing beads, tend to interact with the surface adsorption sites. Depending on the HPLC mode, the different types of the adsorption forces may be included in the retention process: Hydrophobic (non-specific) interactions are the main ones in reversed-phase (RP) separations.

Dipole-dipole (polar) interactions are dominant in normal phase (NP) mode. Ionic interactions are responsible for the retention in ion-exchange chromatography. All these interactions are competitive. Analyte molecules are competing with the eluent molecules for the adsorption sites. So, the stronger analyte molecules interact with the surface. The weaker the eluent interaction, the longer the analyte will be retained on the surface. SEC (size-exclusion chromatography) is another case. It is the separation of the mixture by the molecular size of its components, the bigger the molecule, lesser the chances for it to be retained. In HPLC the mobile phase flow by using a high-pressure pump through a stationary phase composed of small particles of 2-5 micrometer size range. HPLC is the method of choice for the separation of non volatile organic compounds such as hydrocarbons steroids, pesticides, strongly polar and ionic compounds such as amino acids, nucleic acids high molecular weight compounds such as synthetic polymers and biopolymers such as polypeptides, proteins, polynucleotide’s, carbohydrates and thermo labile and decomposable compounds such as terpenoids.

Method of Assay Using Hplc:

The calibration curve or standard curve was prepared after the aspirin content was determined in tablets from different pharmaceutical companies. For the preparation of the calibration curve, the external standard aspirin usp reference standard was used, and the stock solution was prepared, from this stock solution, different dilutions (0.0mg/ml, 0.lmg/ml, 0.2mg/ml, 0.3mg/ml, 0.4mg/ml, 0.5mg/ml and 0.6mg/ml) were made which are then injected into HPLC to obtain the (Aup) of each concentration.

The chromatographic procedure is carried out by using:

  1. Stainless steel column with a dimension of (250 x 4.6mm) that contains packing L1 C18. (ODS).
  2. Diluent: a mixture of acetonitrile & formic acid (99: l) was prepared.
  3. Mobile phase: 2 gram of sodium heptanesulfonate in a mixture of 850 ml of water and 150 ml of acetonitrile was dissolved, and adjust with glacial acetic acid to pH 3.4.
  4. Operate in mode LPG.: - Flow rate 2ml/min. - Run time 5min. - Injection volume 20µl. - Detector wave length 280nm (UV-Visible detector).

Preparation of standard solution and calibration curve:

Accurately 25 mg of Aspirin, Reference standard USP was weighed. The weighed powder was dissolved in 25 ml volumetric flask containing solvent as diluents, the volume was completed to 25 ml using the same diluents to form a stock solution with a concentration of 1 mg/mL After that, transfer different volumes from this stock solution in to 10ml volumetric flask and dilute to 10 ml

with diluents phase to prepare solutions of different concentrations. Then each one of these dilutions was injected in to HPLC (4)

Instrument used: Shimadzu LCVP2010C integrated system equipped with quartenary gradient pump, Column used: Kromasil C18 (180 X 4.6mm),5µm Detector used: UV-Vis detector Wavelength: 277nm Mobile Phase: Acetonitrile: Methanol 60:40(v/v)

Results and Discussion

The Rt (Retention time) of aspirin was found to be 4.303.

Spectrophotometry analysis :

is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength It is more specific than the general term electromagnetic spectroscopy in that spectrophotometry deals with visible light, near-ultraviolet, and near-infrared.

in a scanning spectrophotometer is as follows:

[image: ]The light source is shone into a monochromator, diffracted into a rainbow, and split into two beams. It is then scanned through the sample and the reference solutions. Fractions of the incident wavelengths are transmitted through, or reflected from, the sample and the reference. The resultant light strikes the photodetector device, which compares the relative intensity of the two beams. (5)

The mass of acetylsalicylic acid present in the tablets of aspirin could be determined using spectrophotometric analysis. Because acetylsalicylic acid is colorless, it was hydrolyzed to form salicylate, which in turn was reacted with the chromophoric reagent Fe(H2O)6 3+ to form the chromophore. A chromophore is a colored compound that absorbs light with a certain wavelength. Because acetylsalicylic acid and the chromophore have a 1:1 molar ratio, the amount of chromophore detected is directly proportional to the amount of acetylsalicylic acid present. A calibration curve was generated from a standard solution of acetylsalicylic acid. This curve was fit with Beer’s Law, A = εlc

where A is the absorbance (arbitrary units), ε is the molar absorptivity (concentration/cm), l is the path length of the cuvette (cm), and c is the concentration of the iron(III)salicylate chromophore. Beer’s Law is the theory that connects the absorbance of light by a colored solution with the concentration of the chromophore. (6)

Methods of determination by spectrophotometric:

UV-VIS spectrophotometry was used in order to measure the acetylsalicylic acid concentration of the aspirin tablets. Using 0,1 N sodium hydroxide as solvent, the measurements were taken at 299 nm wavelength. It should be noted that the excipients of the tablets do not interfere with the determination.


IR spectra of the starting material salicylic acid and the product acetosalicylic acid (aspirin) are attached. The two substances have many structural features in common, resulting in similar peaks of their spectra. Each has a strong peak near 1689 cm-1 due to stretching of the C=O bond of the acid group [-(C=O)-O-H].

Each also has a large peak near 1605 cm-1 due to a skeletal vibration of the benzene ring. But you can also see the differences. A key difference is acetylsalicylic acid shows two strong peaks in the carbonyl (C=O) stretching region (1650 – 1800 cm-1), because it has two different carbonyl groups — while salicylic acid has only one. (8)

By bear’s lambert law: calibration curve for acetylsalicylic acid (ASA) was generated using a Barnstead Turner SP-350 Plus spectrophotometer. A 0.104 g sample of ASA was hydrolyzed by heating it in 10.0 mL of a 1.0 M NaOH solution. In order to develop the iron(III) salicylate chromophore complex, five samples of the hydrolyzed ASA solution (0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, and 0.5 mL, respectively) were diluted to 25 mL in 0.020 M iron(III) chloride. The absorbance and transmittance of the five solutions were measured at 530 nm

The mass of ASA in commercial pain reliever tablets was determined using spectrophotomeric analysis. Two tablets (from the same container) were separately ground by hand and each hydrolyzed by heating in 10 mL of 1.0 M NaOH. For each tablet, 0.1 mL and 0.2 mL of the hydrolyzed compound solution were diluted to 25mL in 0.020 M iron(III) chloride. The absorbance and transmittance of

the Result and discussion:

The molar absorptivity of acetylsalicylic acid, as determined by a linear fit of the calibration were measured at 530 nm.


The HPLC quantitative analysis is fast and accurate for Aspirin analysis and can be used for routine work. The Rt (Retention time) of aspirin was found to be 4.303.

UV-VIS and FTIR spectra provide useful information about the analyzed samples. Determination of the amount of active substance, carried out by the use of UV-VIS spectrophotometry, showed that there are differences between the content of the active substance stated on the label and the one experimentally determined. The biggest advantage for chemists who use UV-VIS spectrometers is the accuracy of the device

But The main disadvantage of using a UV-VIS spectrometer is the time it takes to prepare to use one. With UV-VIS spectrometers, setup is key. You must clear the area of any outside light, electronic noise, or other outside contaminants that could interfere with the spectrometer's reading.

The calibration curve generated with standard solutions of the chromophore was fit to a linear equation (Beer’s law), which allows for a precise determination of the amount of acetylsalicylic acid in the unknown.


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  4. 25 U3N. The United State Pharmacopoeia and National Formulary.. In.; (2007).
  5. Zhou YJ, Boudreau DM, Freedman AN. Trends in the use of aspirin and nonsteroidal antiinflammatory drugs in the general US populatio. In.: Pharmacoepidemiology and Drug Safety; 2014.
  6. White JaH. R. CHEM 151/152. CA: Azusa, General Chemistry Laboratory Manual Azusa Pacific; 2013.
  7. chemistry doa. the book of instrumental analysis portsaid : faculity of pharmacy ; 2020.
  8. Cruceanu I GV. Determinarea spectrofotometrică în ultraviolet a acidului acetilsalicilic din comprimatele efervescente, : , Farmacia; 1971.

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Determination of Aspirin Using Two Chromatographic Method Using (Hplc or Gc) And Two Spectrophotometric Method.. (2023, Feb 13). Retrieved from

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