Gomez CHE121L

Methyl red is the indicator dye used in this experiment. The maximum absorbance obtained in its acidic form is 520 nm while 420 nm in its basic form. In the determination of molar absorptivities using 10, 25 and 40 ml aliquots; the absorbance increases both in the acidic and basic solution while the volume of aliquots escalates. Buffer solutions were prepared using the Beer’s and Henderson-Hasselbalch’s equation. The isosbestic point appears at near 420 nm which means that there is only one wavelength where two species have the same molar absorptivity. By plotting the pH vs. absorbance of the prepared buffer solutions at two maximum absorbance (acidic and basic), it was deduced that the spectrum of each solution is dependent on the pH range. The obtained pka of methyl red using these data should be compared to the theoretical pKa of methyl red which is 5.05 ± 0.05. However, this is not achieved due to data deficiency.

or conjugate base. while a weak acid dissociates only partially and forms very little H (e. An acid is classified as 3 + + 3 + + + strong or weak depending on the extent to which the molecules of the acid dissociate into H and its anion. The acid dissociation constant. it is understood that the actual species exists as H O . Ka. A calibration curve is specific to a particular substance. Thus strong acids. the dissociated products. is a measure of an acid’s strength. (H and A ) are so strongly favored. when dissolved in aqueous solution. CH COOH. HCl dissociates virtually100% into H and its anion. HIn(aq) In– (aq) + H+(aq) (1) be created by measuring the absorbance of a large number of solutions of known concentration. CH COO ). When enough base titrant is added to the analyte solution the equilibrium expressed in equation 1 will shift toward products. In this experiment. H . It is able to separate the light into colors because each color of light has a different wavelength than the other colors. and must when aqueous H appears in a chemical equation. A good indicator for a specific acid-base titration has an endpoint with a pH at or near the pH of the equivalence point.g. Therefore. An acid is a substance which dissociates to produce hydrogen ions. The pH range of the color change will be observed and compared with the pH of the equivalence point to determine if the indicator is an appropriate choice for each titration.INTRODUCTION Indicators are weak organic acids (HIn) that change color when deprotonated (In-). Weak acid dissociation can be 3 − + represented as a generic reversible reaction: HA(aq) ⇔ H + A where HA is the weak acid and A is the anion. A calibration curve is a graph of absorbance. phenolphthalein indicator will be used for each titration. the H ion. A spectrophotometer separates light into its separate colors. equilibrium constant can be defined: + – (2) The equilibrium constant is called the acid dissociation constant or acid ionization constant. For this reversible reaction. A strong acid dissociates completely in water (e. Different colored substances absorb varying amounts of specific wavelengths of light. Taking the negative log of the Ka results in more easily expressed pKa values ranging from 0 to 14 for weak acids. versus concentration (Beer’s law). H O . Cl ).. For strong acids. A . how much of a particular wavelength of light is absorbed. which is simply a proton. of a + − . So The result is the formation of more of the deprotonated indicator (In ) and a corresponding color change of the analyte solution (the endpoint). 3 + + − + − dissociate into H and the acetate anion. If the undissociated acid (HA) is favored and the acid is weak. immediately combines with water to form the hydronium ion. of HA. K is measurable a and will be much less than one. For weak acids these values are less than 1 and typically so small that they are expressed with scientific notation.. A few drops of indicator added to the analyte solution before the beginning an acid-base titration. Once in solution. only about 3% of the dissolved molecules of acetic acid.g. a spectrophotometer can be Used to measure how much of a substance is present. that [HA] approaches zero and K approaches infinity. The spectrophotometer can shine a narrow band of wavelengths (essentially one specific color) of light on a sample of substance and then measure how much of that light is absorbed by the sample.

concentration (of pure samples) at both wavelengths and determining the slopes of the lines generated. HIn and In. The values for the four  constants can be determined by doing Beer’s Law plots of absorbance vs. wavelength for each of the pure substances. A solution that contains two colored species can also be analyzed using Beer’s Law (A= bc). and 3) the natural ability of the specific substance to absorb light. the value for b will remain the same and the value for  is constant for a specific chemical at a given wavelength of well the material absorbs light b = path length through which the light passes c = concentration of the solution In general. using two different wavelengths of light. do not have values of K associated with them. So. . in a solution with absorbance AHIn and AIn. The final set of measurements will be collected by measuring the absorbance of solutions of the weak acid at different pH’s. but will change at different wavelengths and/or with a different absorbing species. the result will be a straight line with a slope of b and a y-intercept of zero. respectively. it is a A1 = A1HIn + A1In = 1Hin [Hin] + 1In [In] at 1 (5) The constant  does not change with concentration. 2) the distance the light travels through the sample. Mathematically.which there are very few. These equations can be rearranged to allow determination of each concentration. the concentrations of the two species can be determined if two simultaneous equations can be developed that relate the two. [HIn] = ((A12In)  ( A21In)) / ((2In 1HIn)  (1In 2HIn)) (7) [In ] = ((A2 1HIn) – ( A1 2HIn)) / ((2In 1HIn) – (1In 2HIn)) (8) found that the amount of light absorbed by a specific sample depends on three things: 1) the concentration of the solution. then  = b and Beer’s Law for a two component mixture becomes The best wavelengths for the experiment are selected by measuring the absorbance vs. Because the general equation for a straight line is y = mx + b (4) If A is graphed against c. If the sample path length is combined with. the following equation for Beer’s Law would be true: A2 = A2HIn + A2In = 2HIn [HIn] + 2In [In] at 2 (6) There are now two equations that can be used to determine the concentrations of the two different colored unknowns that are present in the solution. Thus an equation can be written relating these things: A=bc (3) where A = absorbance  = molar absorbtivity -. The two species that are being studied in this experiment are the two colored forms of specific indicators that are weak acids. the total absorbance is A = AHIn + AIn . Mathematically. If there are two species. The two unknown concentrations are determined by taking readings on each solution twice. The actual wavelengths are then chosen such that they will maximize the absorbance for each species. at a second wavelength 2.

0. Then the maximum absorbances of the solutions were determined at 20-nm intervals within the 340 – 625 nm wavelength range. After this in order to recognize the pKa of the dye using HendersonHasselbalch equation. In order to identify the molar absorptivities dilute solutions were prepared.01M HCl for the acidic solution while 0. five solutions at various pH values but with constant total dye concentration were prepared. Afterwards. and 40 mL aliquots of the solution these were diluted in a 50 mL volumetric flasks using 0. first these two forms were prepared.05M HCl was used. For the acidic solution of the dye. their absorbanceswere obtained at λHIn and the λIn-. Through obtaining 10. λHIn and the λIn .EXPERIMENTAL MEHOD To obtain the spectra of acid and base forms of the dye. While. 25.05M borax was utilized solution for the basic solution.01M borax solution for dilution of basic solution. . 0. It was followed by obtaining 10 mL of the original dye solution and placing it into a 100 mL volumetric flask. Then it was diluted to the mark with the buffer solution. The absorbances of the six solutions were measured at the wavelengths of maximum absorption.

003 -0.118 Basic Dye Solution λ (nm) Absorbance 380 0.004 -0.048 415 0.019 0.001 -0.129 0.126 535 0.045 0.042 0.075 0.126 515 0.128 520 0.RESULTS Table 1.077 0.123 540 0.037 390 0. Wavelength and Maximum Absorption of Acidic Dye and Basic Dye Solution (With 20 nm intervals) λ (nm) 380 400 420 440 460 480 500 520 540 560 580 600 620 625 Acidic Dye Solution 0.048 0.034 0.003 Maximum Wavelength of Absorption for Acidic Dye Solution: 520 nm Maximum Wavelength of Absorption for Basic Dye Solution: 420 nm Table 2A.008 0.042 0.034 385 0.001 0.110 0.001 Basic Dye Solution 0. Wavelengths and Absorbance of Acidic DyeTable 2A.003 0.118 0.046 410 0. Wavelengths and Absorbance of Basic Dye Acid Dye Solution λ (nm) Absorbance 505 0.050 0.129 525 0.119 510 0.050 420 0.044 400 0.128 530 0.050 .021 0.003 -0.003 -0.011 0.027 0.045 405 0.004 0.004 -0.040 395 0.

08 0.06 0. Determination of Molar Absorptivities Table 3B.026 40 mL 0. Determination of Molar Absorptivities Acidic Dye Solution λ (nm) 10 mL 25 mL 45 mL 520 0.027 0.02 0 0 100 200 300 400 500 600 700 Wavelength Figure 2.06 0.02 0.05 Absorbance 0.14 0.067 0. Wavelength vs.Table 3A.01 0 -0. Absorbance graph (Basic Dye Solution) Absorbance of the basic solution 0.1 0.011 25 mL 0. Wavelength vs.04 0.107 Basic Dye Solution λ (nm) 420 10 mL 0.03 0.01 0 100 200 300 400 500 600 700 Wavelength . Absorbance graph (Acidic Dye Solution) Absorbance of the acidic solution 0.04 0.12 Absorbance 0.043 Figure 1.

8 5. pH vs.1 0.4 4.06 0.04 0.Figure 3.03 0.8 5.6 pH 6 6.06 0. pH vs. Wavelength vs.04 0.14 0.12 0.02 0.05 0.04 Acidic 0.02 Wavelength Basic Table 4A.02 0 4.12 0.4 4.01 0 4.06 0.08 Absorbance 0.4 0. Absorbance graph (Acidic and Basic Dye Solution) 0. Absorbance At maximum Absorbance of Basic Form 0.4 .6 pH 6 6.02 0 380 400 420 440 460 480 500 520 540 560 580 600 620 625 -0. Absorbance At maximum Absorbance of Acidic Form Table 4B.1 Absorbance Absorbance 0.08 0.

Figure 4. Absorbance of acid and basic form of methyl red at two wavelengths .

The pH of these buffers force methyl red to distribute itself somewhat evenly between the two colored forms.8. The wavelength at which the absorbance is greatest needs to be determined for the reason that the spectrophotometer is more sensitive to absorbance changes at this wavelength. or pKa. since the Ka value for acetic acid is in the same range as the Ka' value for methyl red. buffer solutions with pH 4.1 M were determined first. Based on the data in Table 1. and 6.DISCUSSION After the preparation of the acidic and basic solutions. it was found out that the amount of volumes were very small making it very difficult for the acid and the base to be pipette. then from this the moles of the acid which is acetic acid and the base which is sodium acetate can be determined as well. their molar absorptivies were obtained by using the maximum absorbance of the acidic and basic dye solution. it was prepared by mass with the help of analytical balance and diluting both the acid and the base to 250 ml with distilled water. In order to be prepared. one equation preparing the buffer solutions. 4. by varying the pH and measuring the ratio [In–]/[HIn . So instead preparing the buffer solutions by volume. The absorbance of mixtures is the sum of the separate absorbencies. after calculating the appropriate volumes for the different pH. Based in this. In this experiment we will determine this equilibrium constant. However. We need to set up two equations in two unknowns. which is moles/Liter.7. the molar absorptivity is directly proportional to the absorbance. The absorption of light is governed by the Beer-Lambert Law. Methyl red (4-dimethylaminobenzene2’-carboxylic acid) is a commonly used indicator for acid-base titrations. In the case of the dilute solutions. By following the change in absorbance as a function of pH of the prepared buffer solutions we will determine the acid dissociation constant. This can be explained through Beer’s law from equation 3. The ratio [In–]/[HIn] at different pH values and at . Using the of acetic which is 4.6. Absorbance graph of basic dye solution tends to lean on the left ro to the large presence of its basic forms. The capacity of the material to absorb light or its absorptivity increases as its absorbance increases as well. From Figure 2 and 3.4. the Wavelength vs. From the data gathered in Table 3A and 3B. concentrations are typically measured at maxima in the absorbance spectra because this is where the absorbance changes least with changes in wavelength.4 were prepared. which is 520 and 420 nm respectively. their wavelength at maximum absorbance. pKa'. We will use acetic acidacetate buffers to control the pH. 5. The Henderson-Hasselbalch equation which is [ [ ] ] constant total dye concentration of 0. their volumes must be known by using the equation for molarity. Absorbance graph of acidic dye solution tends to lean on the left due to the large presence of acidic forms while in the Wavelength vs. one can see that the absorptivities of the dilute solutions by using the maximum absorbance in acidic solutions are higher than the basic solution. Although a substance will have an extinction coefficient at every wavelength. the maximum wavelength of absorption for acidic dye solution is 520 nm while 420 nm for the basic solution. was used in The best wavelengths to choose for the analysis are where one form absorbs strongly which is the maximum absorbance.

the absorbance increases as the pH increases using the maximum absorbance at basic conditions. The appearance of an isosbestic point is evidence that only two species are involved. the wavelength of light. By looking at Table 4A. Especially for infrared spectrophotometers. UV/Vis spectroscopy is routinely used in the quantitative determination of solutions of transition metals and highly conjugated organic compounds. For an unknown solution.for each wavelength. Within these ranges of light. Call the two wavelengths and . There are also spectrophotometers that use arrays of photosensors. one can see that the absorbance decreases as the pH increases using the maximum absorbance at acidic conditions. or more specifically. like polished glass. At the isosbestic point the total absorbance of a solution of the two ions is independent of their relative concentrations but is dependent only upon the total dye concentration. Equilibrium constants involving ionic species are especially sensitive to ionic strength. The activity of all the species in solution are a function of the ionic strength. spectrophotometers use a monochromator containing a diffraction grating to produce the analytical spectrum. Historically. In this experiment from Figure 3. A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the color. the isosbestic point is near 420 nm. While in Table 4B. The spectrophotometer quantitatively compares the fraction of light that passes through a reference solution and a test . The spectral changes are explained in terms of shifts in equilibria between different molecular and ionic species in the solutions.1 An isosbestic point is defined as the wavelength where two species have the same molar absorptivity. The molar absorbance coefficients are determined from standard solutions that contain one component alone. there are spectrophotometers that use a Fourier transform technique to acquire the spectral information quicker in a technique called Fourier Transform InfraRed. calibrations are needed on the machine using standards that vary in type depending on the wavelength of the photometric determination. and . This equations provide two equations in two unknowns. Spectrophotometers are most commonly used for the measurement of transmittance or reflectance of a solution or transparent material. Methyl red has a pKa of 5. The ionic strength is a measure of the total ion concentration in solution. the absorbances at the two wavelengths.2500nm using different controls and calibrations. are determined and then the two equations are solved for the unknown concentrations [ ] and [ ] at each given pH. The two measurements then provide two simultaneous equations with two unknowns: [ ] [ ] [ ] [ ] Spectrophotometry involves the use of a spectrophotometer. However they can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 250nm .

.01M. This causes a deviation from the linear relationship because the extent of interaction depends on concentration. is the extinction coefficient. the above equation becomes: Atot = A1+A2+. such that: A = εbc where A is the absorbance. An output wavelength is selected and beamed at the sample. b is the path length (normally 1 cm) and c is the concentration of the absorbing species.t In short. ε. Beer's law is successful in describing the absorption behavior of dilute solutions. A fraction of the monochromatic light is transmitted through the sample and to the photodetector. and the transmittance value for this wavelength is then compared with the transmission through a reference sample.. so the absorbancies of all other substances are recorded relative to the initial "zeroed" substance. Discrete frequencies are transmitted through the test sample. which diffracts the light into a "rainbow" of wavelengths and outputs narrow bandwidths of this diffracted spectrum.+An = ε1bc1 + ε2bc2 + . Beer's law applies to solutions containing one or more absorbing species. . Dilute solutions must therefore be used. if there is no interaction between the various species in the solution. Then the photon flux density (watts per metre squared usually) of the transmitted light is measured with a photodiode or other light sensor. 3. The light source shines through a monochromator. A similar situation can occur when the concentration of the absorbing species is low compared with the concentrations of other species. Light from the source lamp is passed through a monochromator. the concentration of the absorbing species and the path length. is a linear relationship between absorbance and the concentration This is not generally the case.. In the case of a solution containing n species which absorb. Many spectrophotometers must be calibrated by a procedure known as "zeroing.+ εnbcn Beer's law in the case of a fixed path length. b. Beer's law relates the absorbance. This can alter a species' ability to absorb a given wavelength of radiation. The effects of these molecular interactions become negligible at concentrations below 0. One of the fundamental assumptions in the derivation of the law is that the average distance between atoms is large enough such that the charge distributions of neighboring atoms or molecules are not affected by those of its neighbors. the sequence of events in a spectrophotometer is as follows: 1. 2." The absorbancy of a reference substance is set as a baseline value. The spectrophotometer then displays % absorbancy (the amount of light absorbed relative to the initial substance). ε. and extinction coefficient.solution..

Such wavelength changes will occur if either (i) the molar absorbtivity of the precursor changes or (ii) the fraction of the precursor that is converted to multiple products changes.[2] The reason for this is that it would be very unlikely for three . creating the impression that the isosbestic point is 'out of focus'. and the analytical concentration remains constant. the isosbestic point corresponds to a wavelength at which these spectra cross each other. or by using absorbance and keeping the same molar concentration for both species). Thus. deviations can be minimized by using a relatively flat part of the absorption spectrum such as the maximum of an absorption band stray light When an isosbestic plot is constructed by the superposition of the absorption spectra of two species (whether by using molar absorptivity for the representation.The linearity of the Beer-Lambert law is limited by chemical and instrumental factors. A pair of substances can have several isosbestic points in their spectra. regardless of the extent of reaction (or the position of the chemical equilibrium). epsilon(Precursor)(M) is the molar absorbtvity of the precursor. so that it is often assumed that an isosbestic point occurs only when the precursor is quantitatively converted to a single product. If a third one is partaking in the process the spectra typically intersect at varying wavelengths as concentrations change. In the latter case. other ratios than one to one are possible. and epsilon(Product)(M) is the molar absorbtivity of the product. f = epsilon(Precursor)(M)/epsilon(Product)(M). or that it will shift as conditions change. the isosbestic wavelength and molar absorbtivities of the precursor and product can be used to calculate the fraction of the precursor that is converted to products from the relationship. the absorbance of the reaction mixture at this wavelength remains invariant. The requirement for an isosbestic point to occur is that the two species involved are related linearly by stoichiometry. such that the absorbance is invariant for one particular wavelength.01M) due to electrostatic interactions between molecules in close proximity scattering of light due to particulates in the sample fluoresecence or phosphorescence of the sample changes in refractive index at high analyte concentration shifts in chemical equilibria as a function of concentration non-monochromatic radiation. This occurs because the two substances absorb light of that specific wavelength to the same extent. An isosbestic point is observed in overlaid spectra when a chromophoric precursor is converted to a product with a different spectrum. Causes of nonlinearity include:        deviations in absorptivity coefficients at high concentrations (>0. The presence of an isosbestic point typically does indicate that only two species that vary in concentration contribute to the absorption around the isosbestic point. We show experimentally and by computer simulations that more complex reactions also exhibit isosbestic points and that the wavelength of the isosbestic point may change. When a 1-to-1 (one mole of reactant gives one mole of product) chemical reaction (including equilibria) involves a pair of substances with an isosbestic point. where f is the fractional conversion.

bromothymol blue (325 and 498 nm) and congo red (541 nm). isosbestic points are used as reference points in the study of reaction rates. it becomes a very reliable reference. In chemical kinetics. and so. to verify the accuracy in the wavelength of a spectrophotometer. regardless of its saturation. . Oxyhaemoglobin and deoxyhaemoglobin have isosbestic points at 590 nm and near 800 nm. as a quality assurance method. The wavelength of the isosbestic point determined does not depend on the concentration of the substance used.compounds to have extinction coefficients linked in a linear relationship by chance for one particular wavelength. as the absorbance at those wavelengths remains constant throughout the whole reaction. Isosbestic points are also used in clinical chemistry. This is done by measuring the spectra of a standard substance at two different pH conditions (above and below the pKa of the substance). The standards used include potassium dichromate (isosbestic points at 339 and 445 nm). Isosbestic points are used in medicine in a laboratory technique called oximetry to determine hemoglobin concentration.

the ratio [In–]/[HIn] may be determined spectrophotometrically. From the data gathered in Table 3A and 3B. The color of these methyl red solutions should vary from the acidic color to the basic color. using several concentrations to determine whether Beer’s law is obeyed. The pka of methyl red was not able to obtain because to determine the ionization constant of methyl red. Figure 4 is an example of absorbance of acid and basic form of methyl red at two wavelengths. and at the other. The absorption spectra of methyl red in acidic and basic solutions are determined. These two wavelengths. Like in his experiment. it was not able to do in his experiment due to the lack of data gathered. The method is a simultaneous photometric determination. λIn– and λ HIn. The isosbestic point only appears at near 420 nm which means that there is only one wavelength where two species have the same molar absorptivity. however. yellow if it is above 6. Methyl red is a weak organic acid which can be used as an indicator in the pH range of 4.8 to 6.0 and a mixture of both if 4.0.0. the situation is reversed. are chosen so that at one. - . The absorbancy indices of [In–] and [HIn] are determined at both of these wavelengths. the acid dissociation constant of the indicator dye based on the maximum absorbance and molar absorptivities of the acid and base forms of the dye. Since the two forms of methyl red absorb strongly in the visible range. we can verify that a solution of methyl red will be red if the pH is lower than 4.8< pH > 6. the acidic form has a very large absorbancy index compared with the basic form. The ionization constant may be calculated from measurements of the ratio [In–]/[HIn] at known pH values. Spectrophotometer was used to determine the pKa of theindicator solutions.present in solution must be obtained as a function of pH at different wavelengths. or by an analogous procedure for indicators for oxidation-reduction titrations. This method is used to determine concentrations of various chemicals which can give colors either directly or after addition of some other chemicals. From the prepared solutions done in this experiment. we can validate that molar absorptivity is directly proportional to the absorbance. the relative amounts of HIn and In. This method is useful for studying dyes for use as indicators in acid. methyl red. The use of a spectrophotometer to determine the extent of absorption of various wavelengths of visible light by a given solution is commonly known as colorimetry.base titrations.8. using Beer's Law and the Henderson .Hasselbalch equation to determine the pKa of the indicator. Maximum absorbance was used because the wavelength at which the absorbance is greatest needs to be determined for the reason that the spectrophotometer is more sensitive to absorbance changes at this wavelength. and two wavelengths are selected for analyzing mixtures of the two forms.CONCLUSION Absorption Spectroscopic methods of analysis rank among the most widespread and powerful tools for quantitative analysis.

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