Determining Concentrations with Spectrophotometer

Determining Concentrations with Spectrophotometer To learn how to make a spectrophotometer accurately and interpret the data recorded to construct a graph and obtain a standard curve using excel. To learn how mathematical calculations of the absorbance readings and of unknowns to get the standard curve of a concentration value from the curve. Method: Refer to Proc 2048 Biochemical Engineering Lab Manual Exercise 1- The Spectrophotometer Absorbance readings of Methyl Orange and Bormophenol Blue were recorded for a range of wavelengths from 400 to 700nm intervals, zeroing the apparatus with a distilled water blank after each change in wavelength. Exercise 2: Determination of Glucose Concentration Each of the standard glucose solutions and the unknown solutions were tested in spectrophotometer using a wavelength of 580nm and absorbance readings were taken for each. Exercise 3- Determination of Yeast concentration Similarly to the glucose experiment absorbance readings of different standard and unknown yeast concentrations were recorded at a wavelength of 600nm. Unknown solutions U and V were also diluted to a 1:2 ratio with distilled water as the concentrations are too high and fall outside the acceptable absorbance range. Calculations: Unknown glucose calculations y = 0.1836x 0.008, Solve for x gives Subbing in absorbance values for each unknown glucose solution gives: Unknown yeast calculations y = 0.557x + 0.003 Solve for x gives Subbing in absorbance values for each unknown glucose solution gives: U and V need to be multiplied by 2 after calculation as they were diluted in a 1:2 dilution Discussion: In exercise 1 different wavelengths was used and the absorbance in the graph increasing then decreases and then slightly increasing. The colour contributes to where the max absorbance occurs and the concentration affects the intensity of the peaks. For instant the parts blue and the parts yellow light is a green colour. This is the light that we see, and therefore the wavelengths of light to pass through the absorbance with the minimum in terms of all other waves of light absorbance higher. Spectrum analysis of pure sugar solution would be impossible for any absorbance that can happen is the solution to be transparent and any suspended particles. This means that solutions need glucose to the reaction with 1 ml of 3.5 acid Dinitrosalicylic (Domain Name System) to form amino 3, 5 Nitrosalicylic acid, a compound color absorbs light strongly in all parts of 580nm. This enables us to use spectral analysis to determine the focus. This applies only if the intensity of the color of the product is directly proportional to the concentration of the reactants. In this case, glucose concentration is directly proportional to the amount of amino 3, 5 Nitrosalicylic acid producers such as the Stoichiometry of the reaction is 1:01, and most of this focus is not to reach a balance It is important that the blank or zero concentration used for this experiment is not just distilled water but 1ml of DNS and distilled water made up to the same volume as the other samples, as the unreacted DNS in our glucose solutions is contributing to the colour of the solution as well as the 3-Amino,5-Nitrosalicylic Acid. In exercises 3 yeast is basic in terms of chemistry is based on the physics. By increasing the concentration, the absorbance will increases in yeasts solutions however they are not coloured but they are block and scatter, so some the light will not go through them. And this because we are dealing with suspended particulate matter, and not resolved ions. Blot out the light commensurate with the focus so that we can find the concentrations of unknown values of absorbance. It is important to shake well before taking the sample absorbance reading such as yeast, particles and settle to the bottom, that mean if we are not shaken them, so they will give us a lower absorbance reading. Questions Exercise 2 The cuvettes have different surfaces for two reasons. The frosted ridged sides are so no light escapes out the sides of the cuvette giving a false reading. The other reason they have 2 different sides is so that you dont handle the transparent sides directly as oils or dirt from your fingers could increase the absorbance and give inaccurate results Particles in solution (just like in the yeast experiment) affect the absorption reading by blocking or deflecting light away from the detector therefore the particles in a coloured would increase the absorbance and give inaccurate results, unless the concentration and size of the particles is constant with all tests conducted then it would not affect the calculated results. A standard curve in spectrophotometric analysis is a linear trendline that fits through your experimental data. It is calculated by measuring absorbance readings at a range of different concentrations and plotting them against each other. A linear regression done my excel or other means is calculated for the points and an equation in terms of absorbance (Y) and concentration (X) is formed and you can use this equation to calculate unknown concentrations from absorbance readings. Exercise 3 The cuvettes have different surfaces for two reasons. The frosted ridged sides are so no light escapes out the sides of the cuvette giving a false reading. The other reason they have 2 different sides is so that you dont handle the transparent sides directly as oils or dirt from your fingers could increase the absorbance and give inaccurate results Firstly dilute the dye to an adequate concentration with distilled water .Find the maximum absorbance of the diluted dye by testing absorbances at a range of different wavelengths ensuring you zero with distilled between each wavelength. Take some of the dye and dilute it with distilled water to about 6 8 different concentrations i.e. 1:100 à ¯Ã†â€™Ã‚   1:10, depending on what absorbance readings you get adjust the dilutions to fit in a range of 0 à ¯Ã†â€™Ã‚   0.8 as that is where the Beer-Lambert Law applies. Using the max absorption wavelength you would then prepare a standard curve for the dye by measuring absorbance of each of the diluted concentrations. Plot the absorbance vs concentration and use a linear regression to form an equation. Take a sample of the waste water and filter off any suspended particles to increase the accuracy of the absorbance reading. Finally measure the absorbance of the sample diluting accordingly if absorbance is not in the required range. Using th is absorbance value in your standard curve equation calculate the concentration and multiply by your dilution factor if the sample was diluted. Assuming you already have a standard curve and equation for the nitrate complex for a range of 0 à ¯Ã†â€™Ã‚   1.5 mg/L. All you need to do is dilute your sample from your suspected 55 mg/L to fall in the concentration range of 0 à ¯Ã†â€™Ã‚   1.5 mg/L. So a 1:50 or a 1:100 dilution would be enough to give you 1.1 mg/L or 0.55 mg/L respectively. Then the sample is ready for spectrophotometer analysis. Conclusion: All of the yeast and sugar were obtaining accurate test results with the value of R2 (0.99) for each of the trend lines and graphs. 99% accuracy meaning that lines fits for most the points. The process was a good knowledge to learn and it is useful to understand the Beer-Lambert law and his applications.

#15 Synthesis of Ethyl Salicylate from Salicylic Acid

#15 Synthesis of Ethyl Salicylate from Salicylic Acid ____________________________________________________________ _______________ Purpose: Salicylic acid is converted to the liquid ethyl salicylate by addition of ethanol. Yields of salicylic acid (week 1) and ethyl salicylate will be found, as well as the overall yield of the 2-step conversion of acetyl salicylic acid to ethyl salicylate. Introduction: Esters are formed from the reaction of acids and alcohols: O O || || R-C-OH + HO-R’ > R-C-OR’ + H2O cid alcohol ester water The pleasant smelling oil of ethyl salicylate is made by reacting salicylic acid (week 1) with excess ethanol. An acid catalyst is also needed. salicylic acid (SA) ethanol ethyl salicylate (ES) The overall yield is determined from multiplying yields of the two reactions: 1. ASA + HCl(aq) >SA and 2. SA + ethanol > ES + water Apparatus The reaction takes place by refluxing (gentle boiling) in a 100-mL round bottom flask. This time the ice-cooled conde nser is essential, otherwise the ethanol reactant (b. . 79? C) would evaporate before the reaction was over. Melting points of acetyl salicylic acid and salicylic acid (dried from week 1) are taken with MelTemp devices. If you have never used one your instructor will demonstrate. MelTemp C OH O OH H OC H2CH3 H + C OH O + OCH2CH3 + H2O2 Procedure Part A: Esterification of Salicylic Acid with Ethanol 1. Before starting remove a spatulaful (that will be < 0. 1 g) of SA (salicylic acid) and place in a labeled plastic vial to use for melting points. 2. In a 100 mL round bottom flask, mix at least 5 g dry salicylic acid (0. 43 mol) with 20 mL 100% ethanol. The salicylic acid should be weighed out to two decimal places and recorded on the data sheet. The alcohol is in excess. It can be poured from a graduated cylinder, and should not be scaled down. Note: Use more salicylic acid if you have more from the first week. You should have between 7 and 8 g. Adjust amount of ethanol accordingly, s ay to 30 mL. 3. Add approximately 1/2 gram of boric acid catalyst that we use as a substitute for concentrated sulfuric acid in this particular esterification. 4. Place entire assembly on a hot plate and heat until the alcohol is boiling. A setting of around 3 on the hot plate can be tried and then adjusted up or down as needed. You may need to add more ice. Note: While the refluxing is taking place, you can work on Part B, in which the materials you have already made are characterized by taking their melting points. 5. Reflux for a minimum of 1 hour and 30 minutes or longer as time allows. Cool until the flask is no longer hot, then in an ice bath, until the mixture is at room temperature. Transfer to a clean separatory funnel. 6. Add 10 mL 2M NaOH(aq) . The mixture turns milky as approximately 2-3 mL of ester appears at once as an insoluble bottom layer. Ethyl salicylate is an oily liquid with a density of 1. 1 g/mL. Wait a few minutes until the cloudy emulsion begins to clear up. You can estimate the yield by marking the funnel and measuring an equal volume of water. Record this. You will probably get about 1/2 mL for every 1 gram of salicylic acid. 7. You should be able to recover about 1. 5 mL of the ester oil by separating the layers using a separatory funnel. Allow the bottom layer to pour into a vial. This layer contains mostly ethyl salicylate and should have a very pleasant odor, something like wintergreen. It will also include a little NaOH solution and thus will not be clear and sparkling. If you have enough liquid you can attempt the next step. 8. Dry by mixing with a small amount of anhydrous magnesium sulfate. See your instructor to get help with this. Pour off the clear ethyl salicylate. 3 Part B: Melting Points 1. Measure melting points of the dry acetyl salicylic acid and the salicylic acid using the small samples reserved for this purpose. Record on data sheet and compare with literature values. Part C: Yields 1. The 50 aspirin tablets contained 16. 25 g ASA. To estimate yields in your next 2 reactions, assume the wet ASA you used contained 12 g ASA. Then from the mass of dry salicylic acid obtained find the yield of salicylic acid. 2. Convert the volume of ethyl salycilate to grams using the density, 1. 1 g/mL. Record. Estimate yield of ethyl salicylate from salicylic acid and record, retaining just 2 significant figures. 3. Find the overall yield of the two-step process: Step 1: Acetyl salicylic acid (ASA) to salicylic acid (SA) Step 2: Salicylic acid (SA) to ethyl salicylate (ES)4 Data and Results (yield of ethyl salicylate) Name(s) ____________________________________________________________ ____ Part B: Melting Points Compound Measured Melting Point (? C) Literature Value (? C) Acetyl Salicylic Acid 134 – 135 Salicylic Acid 158 – 160 Yields: Assuming ASA (from extraction of 50 aspirin tablets) = 12 g SA (from hydrolysis of ASA) ______ g Reactant SA esterified: ______ (usually most of the amount above) Approximate volume of ethyl salicylate: _______ mL (density ES = 1. 1 g/mL) Mass ethyl salicylate ________g Maximum g SA: Maximum g ES: Enter amounts where indicated in the table below and find the yields. Reaction Reactant > Product Mass Reactant (g) Mass Product (g) Maximum Mass Product (g) Actual Yield % ( 2 sig. figures) ASA > SA 12 g ASA* g SA g SA SA > ES g SA g ES g ES *Assuming 75% extraction yeild. Overall Yield for 2 steps (product of 2 yields above) : Questions: 1. Recalculate the overall yield for a three-step process including the 75% extraction. 2. The synthesis of a new drug may require as many as 30 steps, some of which produce much less than 90% yields. Comment on this (without doing any calculations). ____________________________________________________________ _______________5 Instructor’s Guide Ethyl Salicylate Part B: Melting Points Compound Measured Melting Point (? C) Literature Value (? C) Acetyl Salicylic Acid 134 134 – 135 Salicylic Acid 159 158 – 160 Yields: Assuming ASA (from extraction of 50 aspirin tablets) = 12 g SA (from hydrolysis of ASA) 7. 3 g Reactant SA esterified: 7. 0 g (usually most of the amount above) Approximate volume of ethyl salicylate: 3. 0 mL (density ES = 1. 1 g/mL) Mass ethyl salicylate 3. 3 g Maximum g SA: 10 g ASA x 1 mol ASA x 1 mol SA x 138 g SA = 9. 2 g SA 180 g ASA 1 mol ASA 1 mol SA Maximum g ES: 7. g SA x 1 mol SA x 1 mol ES x 166 g ES = 8. 4 g ES 138 g SA 1 mol SA 1 mol ES Enter amounts where indicated in the table below and find the yields. Reaction Reactant > Product Mass Reactant (g) Mass Product (g) Maximum Mass Product (g) Actual Yield % ( 2 sig. figures) ASA > SA 12 g ASA* 9. 2 g SA 7. 3 g SA 79% SA > ES 7. 0 g SA 8. 4 g ES 3. 3 g ES 39% *Assuming 75% extraction yeild. Overall Yield for 2 steps (product of 2 yields above): 0. 79 x 0. 39 x 100 = 31% Questions: 1. Recalculate the overall yield for a three-step process including the 75% extraction. 2. The synthesis of a new drug may require as many as 30 steps, some of which produce much less than 90% yields. Comment on this (without doing any calculations). ____________________________________________________________ _______________ 1. Overall yield = 0. 75 x 0. 79 x 0. 39 x 100 = 23% 2. The yield would be very very small, making this an expensive process. 6 Instructor’s Guide Ethyl Salicylate(cont'd) Time: maximum Equipment and Materials: per group Items Number Comment stir/hot plates 1 stir bars 1 00-mL round bottom flasks 1 Weighing papers 1 pack per class Ice 50-mL graduates 1 Top loading balance 2 per class With power supply MelTemps 2 per class thermometer 2 per class For Meltemp m. p. tubes (closed) 50 per class 250-mL beakers 1 for water heating bath Microscale glass clamp 1 (yellow) ice-cooled condensers 1 Ring stand 1 Ring stand clamp 1 separatory funnels 1 boric acid 5 g per class antifungal agent spoon spatulas 1 100% ethanol 1 L per class 2 M NaOH 50 mL magnesium sulfate 10 g per class Epsom salts Safety glasses 1 per student Rubber gloves 1 box per class7 Ideas/ Information We make ethyl rather than methyl salicylate, because methanol is toxic. Under Part A. 3. Note: This experiment was designed to be done using household chemicals. Although Science in Motion can provide concentrated sulfuric acid, we have decided that it is too dangerous to handle. Under Part C. 1. Note: Repeated extractions were done in our lab. They all gave about 75% yields and about 12 g dry ASA. 2M NaOH solution could be prepared by adding solid NaOH to a volumetric flask and then diluting with distilled water to the mark on the flask. Molarity mol/L g NaOH for 1 L solution g NaOH 500 mL solution 2 80 40

United States Senator For Florida - 1668 Words

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