Preparation of Standard Curves. Principle

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1 Preparation of Standard urves Principle Many laboratory tests require the measurement of concentration be evaluated or read in a photometer (colorimeter or spectrophotometer). Since these instruments are capable of only measuring the amount of light being allowed to pass through the cuvette, their readout devices display % of light transmitted or mathematically derived absorbance. ne method of obtaining concentration from % transmittance or absorbance is through the use of a standard curve. For our purposes, standard curves are defined as a graphs with absorption or %T plotted on the Y axis, and increasing concentrations of standard along the X axis. If Beer s Law is followed, the resulting line representing absorbance vs concentration will be straight. A standard curve is constructed after obtaining the %T/Abs readings from a number of solutions of known concentration (standards) used in a reaction or procedure. After the readings are obtained each is plotted on semilog (% transmittance) or linear (absorbance) paper against the corresponding concentration. If the procedure follows Beer's Law, the points plotted will generally lie such that a straight line can be drawn through them. The concentration of controls and other unknowns (patient samples) can be determined by locating their %T/Abs reading on the line, then dropping an imaginary line down from that point to intersect the concentration axis. nce the curve is drawn, a number of things must be considered to determine its acceptability. The majority of the curve s points should be on or close to the line. There could be many reasons for a point not being on the line. If the standards are formed from a series of dilutions, the accuracy of the dilutions must be suspect. alculations of the dilutions and spectrophotometer errores are other possibilities. Whether or not the curve passes through the point of origin (the 0"), varies with the procedure. If Beer s law is followed and the procedure is linear at the lower concentrations, the curve s line generally goes through the zero.

2 Uv/ Vis. Spectrophotometry when a beam of radiation passes through matter, apportion is frequently absorbed; this process involves a transfer of radiant energy to the system, thus electrons of atoms or molecules are excited to higher energy levels. The quantity of the absorbed radiation by a certain species is a function of its concentration. The relationship between energy absorption (Absorbance: A) and the concentration () of the absorbing species is given by: A= ε c l (ε = molar absorbtivity l = path length ) omponents of a Spectrophotometer

3 SPETRPHTMETRI ANALYSIS F ASPIRIN The chemical name for aspirin is acetylsalicylic acid. It is an ester derivative of salicylic acid A colored complex is formed between aspirin and the iron (III) ion. The intensity of the color is directly related to the concentration of aspirin present; therefore, spectrophotometric analysis can be used. A series of solutions with different aspirin concentrations will be prepared and complexed. The absorbance of each solution will be measured and a calibration curve will be constructed. Using the standard curve, the amount of aspirin in a commercial aspirin product can be determined H H 3 (s) + 3H (aq) (aq) + H (aq) + 2H (l) [Fe(H ) ] 2 6 Fe(H 2) H + H First the acetylsalicylic acid is reacted with sodium hydroxide to form the salicylate dianion. Then the addition of acidified iron(iii) ion produces the violet tetraaquosalicylatroiron (III) complex. Procedure Part I / STANDARD URVE 1 Mass 100 mg of acetylsalicylic acid in a 125 ml Erlenmeyer flask. Add 10 ml of a 1 M NaH solution to the flask and heat to boiling. 2 Quantitatively transfer the solution to a 250 ml volumetric flask and dilute with distilled water to the mark. 3 transfer 1,2,3,4 and 5 ml of standard aspirin solution to a (10for group A and 15ml for group B) ml volumetric flask or graduated

4 cylinder. Dilute to the 10 ml mark with buffered 0.02 M iron(iii) chloride solution. 4 Measure the absorbance of each solution starting with lower concentration first with a spectrophotometer set at 530 nm. Use the iron (III) solution as a blank. Record the results on the data sheet. 5 Draw the standard curve using excel program on your computer or ordinary sheet Stock oncentration Absorbance solution ml (mg/ml) after dilution

5 5 Part II: Making a Solution of Unknown oncentration from a Tablet. 1. Place one aspirin tablet (record the brand and mg of ASA as indicated on the bottle) in a 125 ml flask. Add 10 ml of 1 M NaH solution to the flask, and heat until the contents begin to boil and the entire tablet has dissolved. 2. Quantitatively transfer the solution to a 250 ml volumetric flask, and dilute with distilled water to the mark. 3. Pipet a 1.00 ml sample of this aspirin tablet solution to a 10 ml volumetric flask. Dilute to the mark with a 0.02 M Fe3+ solution. Label this solution "unknown," Note : 7g Fel3 in 2 L H2 H.W. 1Explain why the wavelength of 530 nm was used. 2.How did the concentration of your aspirin solution compare to the accepted value? 3. Is it better to buy generic or brand name aspirin? Support your conclusion.

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