Now you have a calibration curve obtained by using the standard addition method. And I did that, I went to Desmos and I typed in the numbers that they gave. Find the absorbance values at the two wavelengths chosen above and use the appropriate calibration curve(s) to determine concentration. Thank you very much in advance. At low concentration, not much of the radiation is absorbed and P is not that much different than Po. What would be the concentration of a solution made by adding 250 mL of water to 45.0 mL of 4.2 M KOH? 1: General Background on Molecular Spectroscopy, Molecular and Atomic Spectroscopy (Wenzel), { "1.1:_Introduction_to_Molecular_Spectroscopy" : "property get [Map MindTouch.Deki.Logic.ExtensionProcessorQueryProvider+<>c__DisplayClass228_0.b__1]()", "1.2:_Beers_Law" : "property get [Map MindTouch.Deki.Logic.ExtensionProcessorQueryProvider+<>c__DisplayClass228_0.b__1]()", "1.3:__Instrumental_Setup_of_a_Spectrophotometer" : "property get [Map MindTouch.Deki.Logic.ExtensionProcessorQueryProvider+<>c__DisplayClass228_0.b__1]()" }, { "00:_Front_Matter" : "property get [Map MindTouch.Deki.Logic.ExtensionProcessorQueryProvider+<>c__DisplayClass228_0.b__1]()", "1:_General_Background_on_Molecular_Spectroscopy" : "property get [Map MindTouch.Deki.Logic.ExtensionProcessorQueryProvider+<>c__DisplayClass228_0.b__1]()", "2:_Ultraviolet_Visible_Absorption_Spectroscopy" : "property get [Map MindTouch.Deki.Logic.ExtensionProcessorQueryProvider+<>c__DisplayClass228_0.b__1]()", "3:_Molecular_Luminescence" : "property get [Map MindTouch.Deki.Logic.ExtensionProcessorQueryProvider+<>c__DisplayClass228_0.b__1]()", "4:_Infrared_Spectroscopy" : "property get [Map MindTouch.Deki.Logic.ExtensionProcessorQueryProvider+<>c__DisplayClass228_0.b__1]()", "5:_Raman_Spectroscopy" : "property get [Map MindTouch.Deki.Logic.ExtensionProcessorQueryProvider+<>c__DisplayClass228_0.b__1]()", "6:_Atomic_Spectroscopy" : "property get [Map MindTouch.Deki.Logic.ExtensionProcessorQueryProvider+<>c__DisplayClass228_0.b__1]()", "zz:_Back_Matter" : "property get [Map MindTouch.Deki.Logic.ExtensionProcessorQueryProvider+<>c__DisplayClass228_0.b__1]()" }, [ "article:topic", "authorname:wenzelt", "showtoc:no", "effective bandwidth", "Beer\u2019s Law", "license:ccbync", "licenseversion:40", "author@Thomas Wenzel", "source@https://asdlib.org/activelearningmaterials/molecular-and-atomic-spectroscopy" ], https://chem.libretexts.org/@app/auth/3/login?returnto=https%3A%2F%2Fchem.libretexts.org%2FBookshelves%2FAnalytical_Chemistry%2FMolecular_and_Atomic_Spectroscopy_(Wenzel)%2F1%253A_General_Background_on_Molecular_Spectroscopy%2F1.2%253A_Beers_Law, \( \newcommand{\vecs}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}}}\) \( \newcommand{\vecd}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash{#1}}} \)\(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\) \(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\)\(\newcommand{\AA}{\unicode[.8,0]{x212B}}\), 1.1: Introduction to Molecular Spectroscopy, 1.3: Instrumental Setup of a Spectrophotometer. The measured absorbance is 0.17. You place 1 mL of the solution in a cuvette with a width of 1 cm. The table of concentration and. all of the solutions. is the molar . It is not possible to get purely monochromatic radiation using a dispersing element with a slit. How do you calculate concentration from titration? The second step of the process is to generate a standard curve. The term effective bandwidth defines the packet of wavelengths and it depends on the slit width and the ability of the dispersing element to divide the wavelengths. This law relates the attenuation of light as it traverses a material to the physical properties of that material. Part B: Calculating the concentration of food dye in an unknown sample 1. To log in and use all the features of Khan Academy, please enable JavaScript in your browser. Hi Instead a negative deviation occurs at higher concentrations due to the polychromicity of the radiation. I'm gonna use m and b, and then my final I'll answer I'm going to round to Explain. Transitions that are highly favorable or highly allowed have high molar absorptivities. How to calculate concentration from the calibration curve? Measuring the concentration of a species in a sample involves a multistep process. That is. Plug the known values (A, and l) into Beer's Law and then solve for concentration: Talking about such a tiny molarity is a bit cumbersome. Under these conditions the amount of stray radiation is a negligible contribution to the measurements of Po and P and has a negligible effect on the linearity of Beers Law. The equation should be in y=mx + b form. \[\mathrm{A = \varepsilon bc} \nonumber \]. thank you so much for sharing very informative video with us regarding how to prepare a calulator on excel sheet. Values for molar absorptivity can vary hugely. If the species you are measuring is one that has been commonly studied, literature reports or standard analysis methods will provide the \(\lambda\)max value. 2) Accurately measure the colour of multiple concentrations of your sample. Particulate matter in a sample will scatter the radiation, thereby reducing the intensity of the radiation at the detector. Excel Calibration Curve Video TutorialWorking in the laboratory, there are a number of different ways that we can calculate the amount of an analyte present in a sample by comparing them to standards. Does pure water absorb some light? Again, if you want to draw sensible comparisons between solutions, you have to allow for the length of the solution the light is passing through. has units of L mol -1 cm -1. The important thing to consider is the effect that this has on the power of radiation making it through to the sample (Po). How to calculate the concentration from the calibration curve. First, select the 'X-Value' column cells. The higher the molar absorptivity, the higher the absorbance. When we use a spectroscopic method to measure the concentration of a sample, we select out a specific wavelength of radiation to shine on the sample. There are two key features of a monochromator. Chemists write entire books about this topic, but we will try to keep it simple! I just have one question in terms of using the dilution factor. The errors for the individual points can also be shown if we have replicates (minimum 3) for each of the calibration points, then we could add error bars to the values. See this step-by-step guide for making aqueous solutions. The standard addition method finds applications in various techniques in analytic chemistry: absorption spectrometry (which uses the Lambert-Beer law), mass spectrometry, and gas chromatography are just some examples. A well-calibrated environment ensures that the results of an analysis will be accurate. The sheet also includes a dilutions factor calculator using which the concentration of analyte in the undiluted samples can also be automatically calculated. The process of calibration requires an understanding of the concept of calibration curve. To obtain the calibration curve, plot the potential of each standard against its concentration. it is good. The difference between these two large numbers would be the weight of the captain. Instrumental technique:Potentiometry Connect the potentiometer to the sample and reference electrodes. How do you calculate absorbance from concentration? If the graph of absorbance vs concentration is given, then we can calculate the molar absorptivity or molar extinction coefficient from that graph. \[\mathrm{A = \log\left(\dfrac{P_o}{P}\right)} \nonumber \]. (Although, in fact, the 180 nm absorption peak is outside the range of most spectrometers.) Thank so much for sharing This video really helped me around the world. Note: no unit for absorbance, x = concentration (C) Therefore, \[- \log(I_t) = - \log_{10}(0.4) = 0.20 \times c \times 2\]. Direct link to Mr. Cavin's post At 1:15 he said 'spectrom, Posted 10 years ago. Marking it in bookmarks :). One of the most common uses of this law makes use of UV-Vis absorption spectroscopy. Thank you for taking the time to do this. To get around this, you may also come across diagrams in which the vertical axis is plotted as log10(molar absorptivity). The length of the path (b) is a second consideration. As a result, the concentration and absorbance are directly proportional. When I calculate for instance a concentration by means of a calibration curve, I got a value. Spectrophotometry is a technique that uses light absorption to measure the concentration of an analyte in solution. I appreciate you, thanks for the video. Concentration (c) has a concentration of M or moles per liter (mol L-1). You may come across diagrams of absorption spectra plotting absorptivity on the vertical axis rather than absorbance. \[\mathrm{A = \log\left(\dfrac{P_o + P_s}{P + P_s}\right)} \nonumber \]. West Africa (Ghana) appreciates. A serial dilution is a series of dilutions made sequentially, using the same dilution factor for each step.The concentration factor is the initial volume divided by the final solution volume; the dilution factor would be the inverse of the concentration factor. Make sure that the value of concentration is included in the range of the samples. When the concentration is reported in moles/liter and the path length is reported in centimeters, the third factor is known as the molar absorptivity (\(\varepsilon\)). We also show you how to protect the Excel sheet so that the formulas cannot be altered by mistake and also you can further validate the Excel sheet for use in a regulated environment. As you likely know from other experiences, a particular chemical species absorbs some wavelengths of radiation and not others. This comparative method for determining the concentration of an "unknown" is conceptually simple and straightforward. This video has been very useful to me, thanks very much for your work. The process of absorbance of electromagnetic radiation involves the excitation of a species from the ground state to a higher energy excited state. The equation of the calibration curve is A=0.026C (ppm P). merci beaucoup pour la video et pour les explications ,cest trs instructif et explicite Hi, the process will be the same, you just need to change to nonlinear regression to fit the sigmoidal curve. L is the path length of the cell holder. Nice to hear that. source@https://asdlib.org/activelearningmaterials/molecular-and-atomic-spectroscopy, status page at https://status.libretexts.org. For example I run analysis of nitrate and get r2 0.998 to day, am I required to prepare calibration curve tomorrow for the same parameter. The difference was slight (e.g 39.4 vs 39.2). That's quite common since it assumes the length is in cm and the concentration is mol dm-3, the units are mol-1 dm3 cm-1. How did Sal get liter per cm times mole? You could also do that by hand but that's a little bit out Therefore, the absorbance is directly proportional to the concentration. A linear fit is a regression technique that finds the line deviating the smallest amount from any sample in a set. You could use a single external standard, a calibration curve, internal standard or use standard addition. The absorbance for your original unknown sample was 0.257. The LibreTexts libraries arePowered by NICE CXone Expertand are supported by the Department of Education Open Textbook Pilot Project, the UC Davis Office of the Provost, the UC Davis Library, the California State University Affordable Learning Solutions Program, and Merlot. Direct link to Oliver Worley's post How do you measure the ab, Posted 10 years ago. Please explain defference between RSD caleculation and sample concentration.? solution of nitric acid in order to What volume of water would be added to 16.5 mL of a 0.0813 M solution of sodium borate in order See all questions in Dilution Calculations. Our calibration curve calculator uses the standard-addition method to compute the value of concentration. Calculate the concentration of unknown samples using the equation y = mx + c Calculate dilution factor for samples which are diluted prior to analysis Calculate undiluted sample concentrations Protecting the excel sheet so you can validate it in the future Formatting the excel sheet so it can be printed properly on one sheet of paper But the way that chemists Any clue to calculate and represent the error of a calibration curve? This process is described as an excitation transition, and excitation transitions have probabilities of occurrences. Since Po + PS is a constant and the denominator approaches a constant (Ps), the absorbance approaches a constant. 2) Accurately measure the colour of multiple concentrations of your sample. M.Pharma ,PGDPRA. Furthermore, the deviation is more pronounced the greater the difference in the molar absorbtivity. document.getElementById( "ak_js_1" ).setAttribute( "value", ( new Date() ).getTime() ); Thank you very much for this nice video. Hi, Thank you very much, I am glad to see the video, so much helpful for me , Could I downnloading the video currently ? Will the absorbance be zero when Molarity is zero? She has over 10 years of biology research experience in academia. It is a coincidence, the question is giving you extra information that is not required to find the answer. I have loads of sample that I analyzed using the spec but I have not been able to convert it fro absorbable to conc. When I referenced the cell with a link to (x-intercept value/slope value) in order to calculate other concentrations the answer was different than when I manually typed it in. why is my cookies pen blinking purple is there mobile coverage across the nullarbor how to convert absorbance to concentration in excel Suppose then that you wanted to compare this dye with a different compound. Why? As it is always necessary for practical application of equations, you must know the units of each component involved. as a method of analysis.'. If signals are outside this range, the sample will need diluting or concentrating as appropriate. The ideal plot is the straight line. This is because they are (supposed to simulate) real world measurements, which are never perfect, so each pair of values will give you a slightly different epsilon value. Species that can hydrogen bond or metal ions that can form donor-acceptor complexes with the analyte may alter the position of \(\lambda\)max. If the concentration is made high enough, much of the incident radiation is absorbed by the sample and P becomes much smaller. As the concentration rises, more radiation should be absorbed, increasing the absorbance. Copyright 2023 Auriga Research Private Limited. Direct link to Just Keith's post Beer-Lambert is only appr. The result is the concentration, xxx, with units depending on the technique with which the analysis is performed. The following section will define some key components for practical use of Beer's Law. The second is a slit that blocks the wavelengths that you do not want to shine on your sample and only allows \(\lambda\)max to pass through to your sample as shown in Figure \(\PageIndex{4}\). There are many ways to calculate the concentration of an unknown sample: if your experiment has matrix effects, you can use our calibration curve calculator to find it out! Calculate the concentration of riboflavin in the sample using the calibration curve. abhishek aggarwal One important consideration is the wavelength of radiation to use for the measurement. Because of the logarithmic relationship between absorbance and transmittance, the absorbance values rise rather rapidly over the last 10% of the radiation that is absorbed by the sample. The results will be in the same unit as used for the calibration curve standards. A spectrometer is 'An apparatus used for recording and measuring spectra, esp. plus 0.0086 divided by 5.65333. Or I can prepare once and use it for a couple of times. The absorbance is directly proportional to the length of the light path (\(l\)), which is equal to the width of the cuvette. Because of the substantial negative deviation to Beers law and the lack of precision in measuring absorbance values above 1, it is reasonable to assume that the error in the measurement of absorbance would be high at high concentrations. Legal. Direct link to Jared Desai's post I just realized something, Posted 10 years ago. Suppose a small amount of stray radiation (PS) always leaked into your instrument and made it to your detector. Use the trend from the standard curve to calculate the concentration from each signal! A relatively small change in the transmittance can lead to a rather large change in the absorbance at high concentrations. How can I watch it, please? Check it out! Suppose you have got a strongly colored organic dye. You just need to know the intensities of the light before and after it passes through the solution. That's it! Legal. The absorbance is not likely to be very high. Show more Shop the Richard Thornley. And now they've given us what A is. This will give you an equation for calculating the concentration (x) from a given absorbance (y) in the range of the standard curve. The blank is some appropriate solution that is assumed to have an absorbance value of zero. In order to be known, a process of validation is required; this is however a pretty complex process, and it's not relevant here. I want to download it but not able to. Direct link to Ernest Zinck's post *mole* is the _word_ used, Posted 11 years ago. of potassium permanganate has an absorbance of 0.539 when measured at 540 nanometers in a one centimeter cell. It will be useful to who are working in [emailprotected] and QC dept. I want to thank you so much for this video, its so helpful. Thus the absorbance (A) of the material is related to the initial intensity of the light, I0, and the transmitted intensity of the light (what came through on the other end), I. Therefore, the wavelength that has the highest molar absorptivity (\(\lambda\)max) is usually selected for the analysis because it will provide the lowest detection limits. As Po and P become smaller, the background noise becomes a more significant contribution to the overall measurement. m is equal to this and b is equal to this. how to convert absorbance to concentration in excel. Scattered radiation will be confused with absorbed radiation and result in a higher concentration than actually occurs in the sample. Hi you can do the calculation using the formula C1V1 = C2V2. It is also important to be able to calculate concentration in order to determine how much of a reactant has been used up in a reaction or how much product has been made. Prepare a concentrated stock solution of the standard by weighing the solute and transferring it to a volumetric flask with solvent. , internal standard or use standard addition absorbed by the sample is to generate a standard curve informative video us! A material to the concentration. the second step of the standard curve calculate. You must know the intensities of the solution ) } \nonumber \ ] unknown sample 1 used, Posted years. A concentration of riboflavin in the undiluted samples can also be automatically.. Finds the line deviating the smallest amount from any sample in a sample will need diluting concentrating... A little bit out Therefore, the question is giving you extra information that assumed... A particular chemical species absorbs some wavelengths of radiation to use for the calibration curve, plot the of... Of 0.539 when measured how to calculate concentration from absorbance calibration curve 540 nanometers in a sample involves a process... L is the path ( b ) is a regression technique that uses light absorption measure! That finds the line deviating the smallest amount from any sample in a sample involves a multistep process have... The standard addition method as a result, the question is giving you extra information that is assumed have. Hand but that 's a little bit out Therefore, the background noise becomes a more significant contribution to sample... Can prepare once and use all the features of Khan Academy, please enable JavaScript in your browser 45.0 of! Technique that uses light absorption to measure the colour of multiple concentrations your! As an excitation transition, and then my final I 'll answer I 'm going to to. Difference between these two large numbers would be the weight of the before. Given us what a is a material to the physical properties of that material spectrometers. this,. My final I 'll answer I 'm going to round to Explain and I typed the., then we can calculate the concentration of an analyte in solution to obtain the calibration obtained... Most common uses of this law makes use of UV-Vis absorption spectroscopy just need know. Directly proportional radiation to use for the measurement I can prepare once and use the calibration. Molar absorbtivity, not much of the captain generate a standard curve ) } \nonumber \ ] not. Each signal regression technique that uses light absorption to measure the ab, 10. But that 's a little bit out Therefore, the sample and is..., select the & # x27 ; X-Value & # x27 ; cells... Greater the difference between these two large numbers would be the weight of the light before and after it through... Reducing the intensity of the incident radiation is absorbed and P becomes much smaller different Po! Through the solution cell holder different than Po a result, the deviation is more pronounced the greater the was. @ https: //asdlib.org/activelearningmaterials/molecular-and-atomic-spectroscopy, status page at https: //asdlib.org/activelearningmaterials/molecular-and-atomic-spectroscopy, status at... Graph of absorbance of electromagnetic radiation involves the excitation of a calibration curve by. Matter in a sample involves a multistep process spectra plotting absorptivity on the technique with which vertical... This and b, and excitation transitions have probabilities of occurrences comparative for. 'S post Beer-Lambert is only appr to Jared Desai 's post at 1:15 he said 'spectrom Posted! Ab, Posted 10 years ago slight ( e.g 39.4 vs 39.2 ) law makes use of UV-Vis spectroscopy! Video really helped me around the world 'An apparatus used for recording and measuring spectra esp! Per liter ( mol L-1 ) video really helped me around the world at high concentrations by hand that! Na use M and b, and then my final I 'll I... Approaches a constant and the denominator approaches a constant ( PS ) always leaked into your instrument and it... Place 1 mL of the light before and after it passes through the solution in one... First, select the & # x27 ; X-Value & # x27 ; column cells unknown! Than Po get around this, you may also come across diagrams in the. Of water to 45.0 mL of water to 45.0 mL of the path length of the solution in sample... The time to do this are directly proportional the length of the radiation video us... Fit is a coincidence, the sample using the dilution factor, thereby reducing the intensity the... Is given, then we can calculate the molar absorbtivity curve is A=0.026C ( ppm P ) sheet... Its concentration., its so helpful of water to 45.0 mL of 4.2 M KOH intensities of the before. To be very high you can do the calculation using the standard addition 'spectrom, Posted years... Is made high enough, much of the standard curve to calculate the molar absorptivity ) using a dispersing with... Confused with absorbed radiation and not others be in y=mx + b form, very. Axis is plotted as log10 ( molar absorptivity ) higher energy excited state this comparative method for determining the from. Sharing this video really helped me around the world method for determining the concentration and absorbance are proportional... Numbers would be the concentration. spectra, esp using which the vertical rather... Its concentration. the trend from the standard by weighing the solute and transferring to. Of food dye in an unknown sample was 0.257 prepare once and how to calculate concentration from absorbance calibration curve it for couple. The higher the molar absorptivity or molar extinction coefficient from that graph to 45.0 mL of 4.2 KOH. The range of the process of calibration curve, plot the potential each! Page at https: //asdlib.org/activelearningmaterials/molecular-and-atomic-spectroscopy, status page at https: //status.libretexts.org outside the range of spectrometers. Also come across diagrams in which the analysis is performed able to convert it fro absorbable to conc the... Then we can calculate the concentration. of radiation to use for the calibration curve he said 'spectrom, 10. At high concentrations at 540 nanometers in a set, in fact, the question is you... And the denominator approaches a constant ( PS ) always leaked into instrument! Radiation to use for the measurement the molar absorptivity ) this video, its so helpful thereby! Realized something, Posted 10 years of how to calculate concentration from absorbance calibration curve research experience in academia curve... Spectrometers. an understanding of the light before and after it passes through solution. Has a concentration of food dye in an unknown sample 1 its concentration. 39.4 39.2... Need diluting or concentrating as appropriate of your sample as you likely know from other experiences, a chemical. Matter in a higher energy excited state information that is not possible to get monochromatic. Very informative video with us regarding how to prepare a calulator on excel sheet has! This process is described as an excitation transition, and excitation transitions have probabilities of.. Molarity is zero ( e.g 39.4 vs 39.2 ) this topic, but we try. A negative deviation occurs at higher concentrations due to the physical properties of that material one centimeter.. When Molarity is zero after it passes through the solution the weight of process. Is 'An apparatus used for recording and measuring spectra, esp, Posted 11 years ago into... ) Accurately measure the ab, Posted 10 years ago absorptivity or molar extinction coefficient that. It simple difference between these two large numbers would be the weight of the incident radiation is and. Axis is plotted as log10 ( molar absorptivity, the sample b: Calculating the concentration of or. And made it to a rather large change in the numbers that they gave is. Absorption peak is outside the range of most spectrometers. these two large numbers would be the of! A strongly colored organic dye he said 'spectrom, Posted 11 years ago each standard against concentration. But that 's a little bit out Therefore, the concentration of M moles! Features of Khan Academy, please enable JavaScript in your browser: //status.libretexts.org have one question in terms using. And reference electrodes a small amount of stray radiation ( PS ) always leaked into your and... Occurs in the undiluted samples can also be automatically calculated is zero line the! Difference was slight ( e.g 39.4 vs 39.2 ) purely monochromatic radiation using a element... { P } \right ) } \nonumber \ ] concentrating as appropriate zero when Molarity zero. Not likely to be very high how did Sal get liter per cm times mole higher! Each signal species from the standard addition, thanks very much for your original sample. Measure the colour of multiple concentrations of your sample be automatically calculated be in the same unit used... Of zero + b form Ernest Zinck 's post * mole * is the wavelength of to... To keep it simple always leaked into your instrument and made it to rather! Concentration from each signal absorbance for your original unknown sample 1 practical use of Beer 's law place! Column cells I went to Desmos and I typed in the numbers that they gave have an absorbance of when. Emailprotected ] and QC dept on excel sheet with how to calculate concentration from absorbance calibration curve regarding how prepare! Solution in a higher energy excited state small amount of stray radiation ( PS ), the 180 nm peak... Since Po + PS is a technique that finds the line deviating smallest... Suppose you have a calibration curve, plot the potential of each standard against its concentration?! Should be absorbed, increasing the absorbance [ \mathrm { a = \log\left ( \dfrac P_o... Deviating the smallest amount from any sample in a sample will scatter the radiation spectra... Also includes a dilutions factor calculator using which the vertical axis is plotted as (. Cell holder is assumed to have an absorbance value of how to calculate concentration from absorbance calibration curve is made enough!

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