"THE DETERMINATION OF CHARACTERISTIC OF COLORIMETRIC INDICATOR DYE USING\r\nABSORPTION SPECTROMETRY\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\nAbstract\r\n \r\nExperimental determination of pKa values is frequently achieved by titrations, in which the end point of titration is measured by pH meter or glass electrode. However, when this method is not possible perhaps due to limitation of resources, spectrophotometric methods (which involve absorbance measurements) are used instead. In this experiment, UV/Vis spectrometer is used as a measuring tool. The effects of pH of buffer solution and concentration of NaCl on Bromocresol purple (BCP) stock dye solution are identified by using Beer-Lambert law, theoretical models and plots of various graphs. \r\n\r\nThe spectrum of dye lies within visible wavelength region. The maximum peaks of the spectra are found to be at wavelength of 432 nm and 589 nm respectively, suggesting that the dye is yellow colour at pH4 and purple colour at pH10. The pKa of the indicator dye is found to be 6.2 and the characteristic of the indicator dye is evaluated and compared with literature. It is also found that ionic strength increases directly proportional to absorbance value at a particular wavelength.\r\n\r\nAim\r\n\r\nThis report aims to investigate and determine the characteristics and properties of a colorimetric indicator dye by using absorption spectrometric method (i.e. UV-Vis spectrometer). The effect of ionic strength on absorption spectra of the dye indicator is also explored and identified.\r\n\r\nObjectives\r\n\tInvestigate the influence of pH ofbuffer solution on indicator dye using UV-Vis spectrometer\r\n\tIdentify the effect of NaCl ionic concentration on the indicator dye using UV-Vis spectrometer\r\n\tApplication of theoretical model found from literature (in this case the derivations of model is given in instruction) and plotting of various graphs in order to explore their relationships, with appropriate critical evaluation of results and error analysis. \r\n\r\nTheory and Background\r\nAtom or molecule comprises of many electrons, in which these electrons contain rotational transition, vibrational and electronic energy andcan be excited from ground state to a higher energy state (i.e. higher energy level of empty orbital) when electromagnetic radiation (i.e. photon) falls on the atom or molecule. The energy of photon that is absorbed by the electron is ‘quantized’ and it can be expressed numerically as: \r\n∆E = hv\r\nTherefore, the study of visible and ultraviolet absorption is essential as it provides information about electronic energy levels of molecules. Spectroscopic technique is a spectroscopic technique that is very valuable in chemical analysis as it can measure absorption of radiation as a function of wavelength or frequency. The wavelength lengths of absorption bands can be used to identify functional groups and correlated types of bond in a molecule. By making appropriate assumptions, absorption spectroscopy can also be used to determine concentration, acid dissociation constant (pKa) and properties of a substance in a solution. \r\n\r\nVisible and ultraviolet absorption spectra are frequently measured using an absorption spectrometer(i.e. UV-VIS spectrometer).The working scheme of many modern UV-VIS spectrometers is based on a double-beam design.[1]\r\n\r\n\r\n \r\n\r\nThe beam splitter (V-shape mirror) splits the 2 beams from the filtered source. One of the beams passes through the reference cell or cuvette (in which deionized water is usually used as a reference) while another beam passes through the sample cell simultaneously. Depending on the substances that are contained in the cells, different portions of beams can bereflected, absorbed or transmitted through the cell.\r\n\r\nIf the photonsfrom the beams match the energy gap of the molecules of substances that are present in the cell, the beam will be absorbed. Otherwise, the beam will be reflected or transmitted through the cell. The reflection and scattering spectra of a material depend on adsorption spectrum (i.e. extinction coefficient) and refraction index. \r\n\r\nThe transmitted beams are then detected by photo-detector. The two outputs are amplified, compared and the ratio between two outputs is displayed by computer.\r\nBeer Lambert Law (linear relationship between Absorbance A with solution concentration C) is the essential working principal of UV spectrophotometer.It be expressed as a measure of the absorbance or %transmittance; however in this experiment, it is expressed as absorbance due to its simplicity.\r\n\"log10\" (\"1\" /\"T\" )\"=εlC\" \r\n\"A=εlC \" \r\nApparatus \r\n10 ml glass pipette; 1 ml glass pipette; pipette pump; 10 ml volumetric flask (or graduated flask) ; 5 ml volumetric flask; UV spectrometer; UV-Vis Cuvette\r\n\r\nMaterials\r\npH 4.0-10.0 buffer solutions; 0.025% Bromocresol purple (BCP) stock solution; 0.01 M, 0.1 M and 0.05M sodium chloride solution (NaCl); distilled water (or deionised water)\r\n\r\nMethods and procedures\r\nIn order to make the best use of time, UV-Vis spectrometer is first turned on as it requires some time to start up. In the meantime, a total of 7 standard solutions (each consists of buffer with different pH) are prepared while waiting for the spectrometer to warm up.\r\n\r\nTo produce the standard solution, 1.0 ml of Bromocresol purple stock solution is pipetted into a 10 ml graduated flask by using 10 ml glass pipette. The remainder solution is then made up to 10 ml (as indicated by the horizontal line in the flask) by pipetting 9.0 ml of pH 4 buffer solutions into the graduated flask. The flask is labelled with a sticker in order to avoid misjudgement with other standard solutions. \r\n\r\nAnother six standard solutions are prepared by repeating the same steps as above using 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 pH buffer solutions respectively. 3 ml of these standard solutions are then transferred into UV cuvette respectively. Another two UV cuvettes are prepared by filling with 3 ml of deionised water. \r\n\r\nOnce the spectrometer is ready, cuvettes (both containing deionised water) are placed in the ‘reference’ and ‘sample’ holders. The start wavelength of the spectrometer is set to 350 nm while the end wavelength is set to 800 nm. `Baseline' icon is clicked for screening. When the measurement is completed, the cuvette in the ‘reference’ holder is retained while the cuvette in ‘sample’ holder is replaced by cuvette which is previously prepared. ‘Start’ button is pressed for screening. This step is repeated until all samples are analysed by the UV spectrometer. All results are recorded and saved. \r\n\r\nTo study the influence of ionic strength on the absorption spectra, 1.0 ml of Bromocresol purple stock solution is pipetted into a 5 ml graduated flask. The remainder solution is then made up to 5 ml by pipetting 4.0 ml of 0.01 M, 0.05 M and 0.1 M of NaCl solution into the graduated flask respectively. These samples are transported to cuvette and are analysed by using UV spectrometer in order to obtain the absorption data. \r\n\r\nResults and discussions\r\nTable 1: Colour of the solutions observed during the experiment is tabulated\r\npH\t4\t5\t6\t7\t8\t9\t10\r\nColour\tPure yellow\tDark yellow\tBrown\tSlight purple\tPurple\tModerate Purple\tDark Purple\r\n\r\n \r\nFigure 1: The spectra of the indicator Bromocresol purple dye as a function of pH (i.e. absorbance of solution as the solution’s pH is varied). \r\nFigure 1 shows that as the solution pH changes from acidic to basic, the spectra evolves from the highest peak at 432 nm (denoted by λ1) to spectra where the peak at about 589 nm (indicated by λ2) dominates. All spectra converge to a common point at about 489 nm. This common point is known as isosbestic point and it is independent of pH due to both forms of the dye indicator (HIn and In-) have the same molar absorptivity at this wavelength.\r\nThis result proves that the bromocresol purple dye is a weak acid that can undergo either protonation or deprotonation, depending on the pH (i.e. concentration of H+) of the solution. At pH 4, the solution mainly consists of Hln, hence the solution appears as pure yellow colour.As the solutions become more alkaline (i.e. increasing concentration of ln-), the solution gradually changes to purple colour. The solution is a dark purple at pH10. This relationship can be indicated by the following expression: \r\nHln (aq)↔H^+ (aq)+〖ln〗^- (aq)\r\n\r\n \r\npH\tλ1\tλ2\r\n4\t0.8777\t-0.0055\r\n5\t0.8934\t0.1313\r\n6\t0.5941\t1.0096\r\n7\t0.1587\t2.3014\r\n8\t0.0540\t2.5593\r\n9\t0.0447\t2.6286\r\n10\t0.0315\t2.5999\r\n\r\nTable and Figure 2: Dependence of absorbance HIn and In- on pH\r\nFigure 2 shows that the dye indicator is in HIn form when the solution is acidic (indicated by blue line). Hence, there is a high absorbance at λ1, but there is a small absorbance at λ2 ¬=589 nm as the concentration of [In-] is low. When the solution is alkaline (i.e. high pH), the dye indicator is in the In- form (indicated by red line), giving a high absorbance at λ2 and minimal absorbance at λ1. \r\n\r\nThe entire spectrum undergoes a transformation as the pH of the solution varies. This results in alterations of the equilibrium position. Decrease in [HIn] corresponds to increase in [In-]. Besides that, Beer-Lambert law states that the absorbance at each particular wavelength is directly proportional to concentration. In other words, a decrease in [HIn] also indicates a decrease in absorbance at λ1, whilean increase in [In-] represents increase in absorbance at λ2.\r\n\r\nThe pH at which the inflection point in both lines occurs is the indicator's pKa. However, in practice, it is not easy to accurately and precisely identify the exact inflection point from Figure 2. To get a more precise pKa value, a theoretical model is derived (in this case, the step by step derivations of the model is given in instruction) and the final equation obtained is given by: \r\n \r\n\r\nA graph of pH versus log ((A_λ1-A_(min λ1))/(A_max⁡λ1 -A_min⁡λ1 ) × (A_max⁡λ2 -A_(min λ2))/(A_λ2-A_min⁡λ2 )) is plotted in order to determine pKa\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\npH\tλ1\r\n\r\n5\t-1.288\r\n6\t-0.232\r\n7\t0.7702\r\n8\t1.5695\r\n9\t1.8137\r\n\r\nTable and Figure 3: Linear plot of derived Henderson-Hasselbach relationship.\r\nThepKais located at the y-axis whenlog ((A_λ1-A_(min λ1))/(A_max⁡λ1 -A_min⁡λ1 ) × (A_max⁡λ2 -A_(min λ2))/(A_λ2-A_min⁡λ2 ))=0. From Figure 3, it can be concluded that the pKa of the dye indicator is 6.2. The evaluation of the indicator dye characteristics is discussed in later section.\r\n\r\n \r\n\r\nFigure 4: The spectra of the indicator Bromocresol purple dye as a function of ionic strength (i.e. absorbance of solution as the concentration of NaCl is varied).\r\nIn figure 4, the solution containing the lowest salt concentration (which is 0.01 M in this case), has the highest peak (in the spectrum) at wavelength of 589 nm. While the solution containing the highest salt concentration (i.e. 0.1 M), has the lowest peak.\r\n\r\nIt can be also observed that, in general, the peak of each solution (with different concentration of salt) shifts very slightly to left and gradually becoming smaller with successive increase in ionic strength of the NaCl solution.\r\n\r\nThe ionic strength of NaCl slightly affects the equilibrium (i.e. protonation or deprotonation) of bromocresol purple dye in the solution. As the ionic strength (i.e. concentration of NaCl) increases, higher concentration of Hln form of the dye present in the solution, resulting in the highest peak at wavelength of 432 nm and the lowest peak at 589 nm due to lower concentration of ln- form in the solution. There is also a likelihood that the solution gradually changes from C21H16Br2O5S to C21H15Br2O5SNa in the presence of increasing ionic strength [2][3]. The UV/Vis spectrometer detects the shielding effects of the NaCl and hence the absorption of the dye is gradually decreasing at 589 nm.\r\n\r\n\r\n\r\nNacl (M)\tlog (NaCl)\tAbs\r\n0.01\t-2\t2.013\r\n0.05\t-1.301\t1.881\r\n0.1\t-1\t1.827\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\nFrom Figure 5, it can be seen that the change is linear with increasing ionic strength when the concentration of the dye remains constant. NaCl concentration increases directly proportional to absorbance value at a particular wavelength, in conformity with Beer-Lamber law. \r\n\r\nIt can be predicted that the ionic strength has similar effects on the buffered solution. In order words, for a particular pH buffer solution, NaCl concentration increases directly proportional to absorbance value at wavelength of 589 nm.[3]. \r\n\r\n\r\nError Analysis\r\nTable 4: The apparatus and its respective systematic errors that have been used while conducting experiment\r\nPipette\tBeaker\tVolumetric flask\r\n(10 ± 0.1) ml\r\n(1 ± 0.006) ml\t(50 ± 5) ml\r\n(100 ± 10) ml\t(10 ± 0.02) ml \r\n(5 ± 0.02) ml \r\n\r\nSystematic errors in preparation of standard solutions of dye\r\n(1 ± 0.006) ml pipette, (10 ± 0.1) ml pipette and (5 ± 0.02) ml volumetric flask are used for preparation of standard solution of dye. The propagation of error in preparing 1.0 ml of Bromocresol purple stock solution and 9.0 ml buffer solutions in the volumetric flask is calculated as below: \r\n(δV/V)^2= (δa/a)^2+(δb/b)^2+(δc/c)^2+ ….\r\n(δV/V)^2=(〖0.006/1.0)〗^2+(〖0.1/9.0)〗^2+(〖0.02/10)〗^2=1.67〖×10〗^(-4)\r\n(δV/V) =0.013\r\n δV = V×0.013= 10 ml×0.013=±0.1 ml\r\n\r\nSystematic errors in preparation of different concentration of NaCl solution \r\n(1 ± 0.006) ml pipette, (10 ± 0.1) ml pipette and (5 ± 0.02) ml volumetric flask are used for preparation of standard solution of dye. The propagation of error in preparing 1.0 ml of Bromocresol purple stock solution and 4.0 ml buffer solutions in the volumetric flask is calculated as below: \r\n(δV/V)^2=(〖0.006/1.0)〗^2+(〖0.1/4.0)〗^2+(〖0.02/5)〗^2=6.77〖×10〗^(-4)\r\n δV = V×0.026 ml=5 ml×0.026=±0.1 ml\r\n\r\nIt can be concluded that all preparation of solutions during the experimental work is subjected to the systematic error of ±0.1 ml, regardless of pH of buffer solution or concentration of NaCl that are being used.\r\nConclusion(Evaluation of indicator dye characteristics and comparison with literature)\r\nTo sum up, the Bromocresol Purple is characterized as a dye indicator that has a pKa of 6.2. The pKa (a method of measuring the strength of an acid) expresses acidity of a particular hydrogen atom in the dye indicator.\r\n\r\nBy comparing with the literature[4], pKa of 6.2 suggests that the dye is a relatively weak acid (due to the fact that the higher the pKa, weaker the acid) which has transitionpH range of 5.2 to 6.8.\r\n\r\nAt a pH below 6.2 (let say pH 4), the dye exists mostly as acidic form of Hln and is slightly less soluble. At a pH above 6.2, (let say pH 10), it exists mostly in its conjugate base form of ln- and is more soluble.\r\n\r\nThe spectrum of Bromocresol Purple lies within visible wavelength region. The maximum peak at wavelength of 432 nm suggests that the dye absorb blue colour of the light. Hence, the complementary colour is yellow; resulting in the dye of solution looks yellow. The maximum peak at wavelength of 589 nm suggests that the dye absorb orange/yellow colour of the light. Hence, the complementary colour is purple which causes the dye in the solution looks purple.\r\n\r\nA common feature of this coloured dye compound suggests that Bromocresol Purplecontains a system of extensively conjugated π-electrons.In essence, this means that it contains some aromatic rings. This property is verified after comparing with literature[4][2]. The structure of the Bromocresol Purple is as shown in Figure 6.\r\n\r\nThe influence of the ionic strength on the ultraviolet–visible absorption spectrum of bromocresol purple is linear (logarithmic). It is also predicted that this influence of ionic strength has similar effects on both non-buffered and buffered solution (i.e. NaCl concentration increases directly proportional to absorbance value at a specific wavelength of a buffered or non-buffered solution).\r\n\r\nIt is predicted that the melting point of the bromocresol purple will increase due to likelihood that it changes from C21H16Br2O5S to C21H15Br2O5SNa as the concentration of ionic strength increases [2]."