BCH4160/2011:Notebook/Brittany's Lab Notebook 2011

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Entry title Solution Equilibrium: Determining the acid dissociation constant of methyl red Overview To prepare a Buffer solution over a range of acidic pH's and basic pH's to test using methyl red indicator dye. This will help determine the acid dissociation constant in methyl red. Materials 50ml centrifuge tubes Base-phosphate Acidic-Citrate 1 pH Indicator Spectrometer Procedure: 1. The spectra of both the acidic and basic forms of methyl red will be examined to determine the optimal volumes of methyl red stock solution and buffer to use in each pH assay. To do this, methyl red stock solution will be added to either phosphate citrate buffer. The spectra of the acidic and basic forms of methyl red (350-600nm) will also be used to determine optimal wavelengths for assessing the amounts of acidic and basic forms of methyl red in solution. 2. A pH meter will be used to determine the pH of the buffer solution after each addition of NaOH. After each pH adjustment, an aliquot of the buffer will be removed from the reservoir and combined with the pre-determined amount of methyl red solution (from step 1) to give samples for spectral analysis. 3. The spectra of solutions at each pH will be measured and recorded using a Vernier spectrophotometer (350nm - 600nm). If necessary, spectral files may be exported as ASCII files for import into Excel for further analysis. Spectra will be used to determine the absorbance of acidic and basic forms of methyl red in each sample, using the optimal wavelengths for each form (determined in step Data analysis: Data analysis will involve creating a series of plots to show the effect of changing pH on the visible spectrum of methyl red. From these spectra, the relative amounts of acidic and basic forms of the indicator may be approximated to calculate a pKa (and Ka) for the dye. To help you with your analysis, I am showing some sample data that I obtained in a study of phenol red, another pH-sensitive dye. 1. Plot an overlay of selected spectra as a function of pH. Label the plot clearly so that the effect of changing pH on specific spectral features is clearly seen. Label the spectral peaks used in quantifying relative amounts of acidic and basic forms of the dye. (You may wish to show the spectra of the acidic and basic forms for clarity.) 2. Calculate the approximate fraction of acidic and basic forms of the dye at each pH using spectral data. 3. Plot the fraction of acidic and basic forms as a function of pH. 4. Plot log([A-] / [HA]) vs. pH. This plot should give a line with an x-intercept equal to the pKa of the dye. Data Figures Notes Spectrometer was being varying and the power went out that day. this forced us to recalibrate which would have subjected our data to slight error with having to recalibrate. References Johnathan Cannon-pHAbsBCH4160_laboratory_090210.docx-located in Blackboard folder QTI PLOT SAMPLE GRAPH Sept. 27, 2011 Overview To Navigate and understand the QTIPLOT applications for plotting data points and using functions to manipulate the graph Materials Computer Procedure: Open QTI Plot application Download sample data points and plot on a scatter plot graph Change fonts, colors, and number scale Add a fit function for the graph (come up with it on your own) Data Figure NOTES The Power went out I made my own function for the curve using laura's graph Bomb Calorimetry First week of experiment rotation The purpose of this experiment is to examine over the temperature change with time. Materials Benzoic acid Oxygen cylinder Isothermal bomb calorimeter 2L volumentric flask 25°C water bath 1mL pipet [edit]Procedure GRAPH Reference to the following procedure: "Bomb calorimetry: The energy of pizza" Stout, Roland P.; Nettleton, Faith E.;Price, Lynn M. J. Chem Educ. 1985, 62, 438-439. contact this project was done with Laura Lee and Jigesh PCR The purpose of this experiment was to learn some of the basics of polymerase chain reactions (PCR) and gel electrophoresis. We analyzed the genotypes of several DNA samples, one obtained from hypothetical crime scene and four from hypothetical suspects. Materials PART 1 Suspect DNA A through D Crime Scene DNA Thermal cycler Part 2 Gel electrophoresis system Agarose 50X TAE buffer Fast Blast™ DNA stain Procedure Purpose of this procedure was to use PCR to amplify DNA from crime scene. Reference "Crime Scene Investigator PCR Basics™ Kit" from Biotechnology Explorer™ by BIO RAD. Results No results were gained from this experiment. Errors were made in gel staining and bands were non-interpretative. Contact Brittany webster; laura Lee Preparation of Lipid Films week 3 The purpose of this protocol was to prepare lipid films for use in future experiments. Materials Materials below provided by Dr. J. Cannon. CHCl3 MeOH Lipid solution Vial [edit]Procedure Add 6mL CHCl3 and 2mL MeOH for a total of 8mL of 3:1 CHCl3:MeOH. Take 2.5mL of CHCl3/MeOH solution and add it to the lipid solution in the vial (will have 2 vials total). Use 1mL of lipid/CHCl3/MeOH solution and place it in a 4mL vial (should have 5 vials total). Dry the vials using a roto-evaporator. Place vial in a desicator overnight to thoroughly dry. Store vials in freeze for future use. Binding Fluorescence: Preliminary Preparations The purpose was to prepare solutions of concentrations 2-30μL in preparation for future binding fluorescence lab. Materials Materials used in this experiment: Lipid films made using protocol on October 20th Peptide (MW = 642.79g) Sodium phosphate buffer (made using [[]] protocol) Fluorescence spectrophotometer [edit]Procedure Preliminary Preparations of Peptide Solutions The purpose of protocol is to prepare peptide solutions of concentrations 2μM, 10μM, and 30μM for future binding fluorescence lab. Add 0.001g peptide to 1mL sodium phosphate buffer. The pH should be ≈ 7.0. Use a UV spectrophotometer to measure the absorbance of the peptide solution at 280nm. Calculate the concentration of the peptide solution using the measured absorbance and Beer's Law. Beer's Law: A=εlc → c = (A÷(εl)) ε=5500 M-1 cm-1 Wavelength = 280 nm l = 0.2cm Once the concentration of the stock peptide solution is known, make 1mL dilute peptide solution in sodium phosphate buffer (2μM, 10μM, and 30μM peptide). Binding Fluorescence Continued The goal of this experiment was to measure the binding fluorescence of a large unilamellar lipid vesicle/peptide solution in order to measure the K of the reaction. Materials Refer back to above section of this experiment. Procedure Preparation of the Lipid Vesicles Add 3mL of the buffer into the pre-made lipid vesicles Vortex for 1 hour Mass of lipids = 0.025j 760.10 g/mol Concentration = 9.39 mM Extrusion of the lipid solution (method used to get unilamellar vesicles) Dilute stock lipid to 5μL, 25μL, and 100μL Controls Obtain a baseline with the following: sodium phosphate buffer peptide solution of all concentrations lipid solution of all concentrations Testing 1:1 dilution of each concentration at 25 °C Total of 9 combinations of lipid-peptide solutions Solution of 5μL lipids with 2μL peptide, 5μL lipids with 10μL peptide solution, and 5μL lipids with 30μL peptide solution Solution of 25μL lipids with 2μL peptide, 25μL lipids with 10μL peptide solution, and 25μL lipids with 30μL peptide solution Solution of 100μL lipids with 2μL peptide, 100μL lipids with 10μL peptide solution, and 100μL lipids with 30μL peptide solution Data & Results Graphs must me redone to upload to this application: Contact Jigesh; Laura ;brittany Enzyme Kinetics Part 1 The goal is to measure the kinetics of the digestion of starch by cellubiase and with and without urea and betaine monohydrate in solution. Materials In this experiment we used: 1.2% starch stock solution 9 molal stock urea solution 10X stock resuspension buffer (pH 5) Distilled water Iodine Cellobiase UV spectrophotrometer [edit]Procedure Refer to Tiffany Byerly's enzyme kinetics protocol Solution Prepartation Prepare X resuspension buffer from original 10X solution (pH 5) Dissolved 16.7 mL original in 150mL distilled water Prepare 1.5% starch soltion Dissolve 1.5g starch in 100mL buffer Prepare 4 molal (mol/kg) Betaine monohydrate Dissovle 2.7g Betaine monohydrate in 5mL buffer Prepare 9 molal of urea Dissovle 2.7g urea in 5mL buffer Creating Baseline Obtain baseline spectrum of 1.2% solution starch and iodine Dissovle 1mL 1.2% starch, 1mL buffer, and 5μL of iodine Note: Final concentraions are 0.6% starch in solution. Measure Absorbance with UV specrophometer Experiment 1 Purpose: Test solution with cellobiase without urea or betaine monohydrate. Add 1mL of 1.2% starch solution, 1mL of buffer and 5μL of iodine to a cuvetter. Shake well to mix. 10μL of cellobiase solution of cuvette. Quickly mix and place in UV spectrophotometer to measure absorbance. Repeate experiment for duplicates. Experiment 2 Purpose: Test solution with cellobiase with .5 molal of betaine monohydrate and without urea. Add 1mL of 1.2% starch solution, 1mL of buffer and 5μL of iodine to a cuvetter. Shake well to mix. Add 0.25mL of betaine monohydrate (for final concentraion of .5 molal) to cuvette. Shake to mix. 10μL of cellobiase solution of cuvette. Quickly mix and place in UV spectrophotometer to measure absorbance. Experiment 3 Purpose: Test solution with cellobiase with 1.0 molal of betaine monohydrate and without urea. Add 1mL of 1.2% starch solution, 1mL of buffer and 5μL of iodine to a cuvetter. Shake well to mix. Add 0.5mL of betaine monohydrate (for final concentraion of 1.0 molal) to cuvette. Shake to mix. 10μL of cellobiase solution of cuvette. Quickly mix and place in UV spectrophotometer to measure absorbance. Experiment 4 Purpose: Test solution with cellobiase with 2.0 molal of betaine monohydrate and without urea. Add 1mL of 1.2% starch solution, 1mL of buffer and 5μL of iodine to a cuvetter. Shake well to mix. Add 1.0mL of betaine monohydrate (for final concentraion of 2.0 molal) to cuvette. Shake to mix. 10μL of cellobiase solution of cuvette. Quickly mix and place in UV spectrophotometer to measure absorbance. Experiment 5 Purpose: Test solution with cellobiase with 3.0 molal of betaine monohydrate and without urea. Add 1mL of 1.2% starch solution, 1mL of buffer and 5μL of iodine to a cuvetter. Shake well to mix. Add 1.5mL of betaine monohydrate (for final concentraion of 3.0 molal) to cuvette. Shake to mix. 10μL of cellobiase solution of cuvette. Quickly mix and place in UV spectrophotometer to measure absorbance. Experiment 6 Purpose: Test solution with cellobiase with 1.0 molal of urea and without betaine monohydrate. Add 1mL of 1.2% starch solution, 1mL of buffer and 5μL of iodine to a cuvetter. Shake well to mix. Add 0.222L of urea (for final concentraion of 1.0 molal) to cuvette. Shake to mix. 10μL of cellobiase solution of cuvette. Quickly mix and place in UV spectrophotometer to measure absorbance.