Background BIOCHEMISTRY LAB CHE-554. Experiment #1 Spectrophotometry
|
|
- Mildred Hardy
- 5 years ago
- Views:
Transcription
1 1 BIOCHEMISTRY LAB Experiment #1 Spectrophotometry CHE-554 In day 1 we will use spectrophotometry as an analytical technique using a known extinction coefficient to assess the precision and accuracy of a common operation in a biological chemistry lab: pipetting. In day 2 we will undertake an experiment wherein we will determine the extinction coefficient of a protein thus permitting direct determination of concentration in the future. We will also use a common indirect method wherein a dye is used in a chromogenic assay. Relevant material is provided Professor in the text Testa in experiment 1, beginning page 15. However, we will use the Bradford reagent instead of Folin-Ciocalteau, we will omit studies of riboflavin and adenine. (We will instead demonstrate a direct protein assay.) (Introductory material beginning on page 3 of the text may also prove useful.) 2 Background! Photometry relates to the study of light.! An experimental tool for producing and measuring a spectrum of light, visible or ultraviolet, is the UV-VIS spectrophotometer.! The UV-VIS spectrophotometer produces incident light and measures the light that passes through the sample (is not absorbed). The machine calculates how much light was absorbed, and presents that to the user.! Solutions absorb at specific wavelengths (energy levels) of light, and this is a function of the material in the solution. Particular materials have a characteristic absorption spectra through a range of wavelengths. Therefore, one can obtain information about a solution by measuring its absorbance.! The absorption of a solution at a specific wavelength also depends on the concentration of sample. Therefore, one can measure the concentration of known material via UV-VIS spectroscopy.! In the visible range, wavelengths of light not absorbed by the sample make up the color of the sample that you see.
2 Theory of absorbance -1 Each photon has a probability γ of being absorbed if it encounters a molecule of dye (absorbing substance). Theory of absorbance -2 # photons absorbed = # photons entering dye x γnaπr 2 C l = # photons entering dye x ζ C l d (# photons) = - # photons x ζ C dl X photons incident (1-γ)X photons transmitted Upon passage through a small amount of solution, the path length is very short: dl (a small change in position l ) 3 γx photons absorbed (γ = here) r If a photon s path passes through a solution with C x NA molecules of dye per L, we consider that a photon affects molecules within a cross-section of area πr 2 and the length of the path through the dye is l (letter l ), then the photon is expected to encounter C x NA x πr 2 x l molecules and have a probability γ C NA πr 2 x l of being absorbed. r is assumed to depend only on the molecule s identity and the wavelength of light. C is the concentration (moles L -1 ). # photons absorbed = # photons entering dye x (γnaπr 2 ) C l l 4 dp= -ζ C dl x P dp/p = - ζ C dl ln(p)-ln(po) = -ζ Cl - - ζ C 0 ln(p/po) = -ζ Cl P/Po = e -ζcl Incident light Po (power at zero thickness of absorber) The number of photons changes by a small amount: dp. l (letter l) ln (P/Po) = log(p/po) log(p/po) = -ζ Cl /2.303 = - εcl, ε=ζ /2.303 transmitted light Pl (power at l thickness of absorber)
3 Theory of absorbance -3 log(io/i) = A = εcl, Eq. 1-7 C is concentration, l is path length, ε is molar extinction coefficient. ε (and therefore A) is a function of the wavelength of the light. If the dye is too concentrated, some molecules may be in the shade of others and not have their expected probability of absorbing a photon. Non-linear regime, Beer-Lambert law no longer holds for high C or long path lengths. Plot A/ Cl = ε The Beer-Lambert Law A = ε c l This equation relates the concentration of the lightabsorbing compound and the path-length of incident light to the absorbance of a solution. A is the measured absorbance of the sample ε is the extinction coefficient, which is a constant that depends on the structure of the material, the wavelength of incident light, and the solvent C is the calculated concentration of the sample l is the length of the path that the incident light travels through the sample A = εcl, slope = εl Units of ε: M -1 cm -1 In these experiments, measure the absorbance using a spectrophotometer and calculate the concentration of sample in solution. 5
4 The electromagnetic spectrum Absorbance vs. wavelength λ (nm) ε (M -1 cm -1 ) nm ! ! in ethanol band II band I
5 Significance of wavelength Transitions between electronic states λ = c/ν, ΔE = h ν, = hc/λ ν is the frequency, λ is the wavelength and c is the speed of light. h is Planck s constant, = 6.6 x J/s λmax is the wavelength with the maximal ε for a given band. It corresponds to the energy of the transition associated with that band. Long wavelength photons carry less energy, shortwavelength photons carry more energy. Longer wavelengths n-π* transitions, mid-wavelenths π-π* transitions. In biochemistry nm, 180 nm, respectively. Eg. N-containing bases of DNA: 260 nm absorbance. Hence the danger of UV light to DNA
6 The visible portion of the EM spectrum Making a measurement Violet: nm Indigo: nm Blue: nm Green: nm Yellow: nm Orange: nm Red: nm Spectrophotometer 11
7 Selecting a wavelength Spectrophotometer Slit Why use absorbance?! It is often a MUCH more accurate way to know concentrations than the weights and volumes used to produce them.! The advantage provided depends on the magnitude of the extinction coefficient (why?)! Accuracy is different from precision (how?) Sample! This is the object of our activities on Day 1. 14
8 Sample standard curve Sample standard curve Sample data set Interpolation or the use of the equation of the line allows determination of the unknown concentration. In this case the unknown falls out of range and requires extrapolation, which is much more dangerous than interpolation. A A 15 C (M) A non-zero intercept may be real, for example due to a reaction with the buffer. 16 C (M)
9 Sample standard curve In this case the unknown falls out of range and requires extrapolation, which is much more dangerous than interpolation. Sample standard curve Determination of the extinction coefficient. A = εc + 0 (with a 1 cm path length) Assuming an intercept 0, the slope is ε. Why do we use the slope instead of just one (C, A) pair? A A 17 C (M) 18 C (M)
10 What we will do Day 1: Validation of techniques and refresher on uncertainties. Bromophenol blue Concentration? c = mass/mw vol Make an illegal measurement, break Beer- Lambert s law. Concentration? c = A/εl? Dilute to A < 1 Concentration? c = A/εl Statistics based on independent repetitions of the dilutions and absorbance measurements. Validation based on comparison with authentic standard solution
11 Day 2, Experiment 1: A chromogenic assay! Non-absorbing compounds can be detected via a reaction that generates a chromophore in proportion to the compound s concentration.! Either a known ε or a standard curve are used to relate the A to the starting compound s concentration. (The standard curve in-essence yields ε).! We will use the Bradford reagent, which is a solution of Coomassie blue G250 in ethanol/phosphoric acid. This is less tricky than the text s recommendation of Folin- Ciocalteau.! The product sheet for Sigma s Bradford reagent is provided on the course web site. We will use a variant of the standard procedure A Bradford Assay! Marion Bradford published and patented the assay. Bradford, M. M. (1976) Anal. Biochem. 72: (This is one of the most heavily cited scholarly articles of all time).! Based on a shift in the absorbance maximum of Coomassie brilliant blue G-250 upon binding to arginine side chain (red form of dye converted to a more blue form).! Two chemical bases for the λmax shift: Acidic dye is added to protein, λmax of the dye shifts from 465 nm to 595 nm. Dye binds to basic and aromatic amino acids especially Arg. Detergents and alkaline phs interfere with the dye s colour shift.
12 Coomassie brilliant blue G-250! At acidic ph, the Ns are protonated, the sulfonates remain ionized, net charge is +1 colour is red.! At neutral ph the Ns are deprotonated, only one is +ve, molecule is an an anion. Molecule is green with ε ~ 43,000 M -1 cm -1.! Binding to protein stabilizes the anion, and produces the blue-green form even when free dye molecules remain cationic (red).! Initially used to dye wool (keratin). - +Arg + R-250 lacks two methyl groups Precautions for Chromophorogenic assays! The reaction must be limited ONLY by the compound to be measured. (Every molecule of compound is counted)! A linear relationship must be demonstrated for the absorbance and the reactant that forms the dye.! Conduct the experiment in such a way that the readings corresponding to unknown samples fall within the reading that make up the standard curve.! If necessary, make dilutions of the unknown. Do this BEFORE conducting the reaction. 23 Structure of Coomassie brilliant blue G Coomassie_Brilliant_Blue 24
13 Day 2, Experiment 2: Direct absorbance measurement on a protein! We will exploit the strong absorbance of UV radiation by tryptophan (Trp) and tyrosine (Tyr) side chains in a protein.! Each protein species has a characteristic 3D structure that places its various Trp and Tyr side chains in unique environments and causes them to have extinction coefficients that vary quite widely.! However if a protein is denatured to a random coil all the side chains are exposed to the medium and behave as if they were all simply amino acids dissolved in that medium Amino acids that absorb strongly in the UV. Garrett and Grisham, 3rd ed. Fig. 4.15
14 A typical protein: Lysozyme UV-absorbing amino acids 27 2ZYP.pdb 28 6 Trp and 3 Tyr.
15 UV-absorbing amino acids! 6 Trp Trp and and 3 Tyr. Tyr.! Some are buried, others are stacked. 29 Denatured protein! In a denaturing medium, the extinction coefficient of the protein at 280 nm can be approximated as the sum of the contributions of the Trps and the Tyrs: εprotein = ntrp εtrp + ntyr εtyr! We will use the protein lysozyme from chicken egg white. the amino acid sequence of this protein is known 1 : LYS VAL PHE GLY ARG CYS GLU LEU ALA ALA ALA MET LYS ARG HIS GLY LEU ASP ASN TYR ARG GLY TYR SER LEU GLY ASN TRP VAL CYS ALA ALA LYS PHE GLU SER ASN PHE ASN THR GLN ALA THR ASN ARG ASN THR ASP GLY SER THR ASP TYR GLY ILE LEU GLN ILE ASN SER ARG TRP TRP CYS ASN ASP GLY ARG THR PRO GLY SER ARG ASN LEU CYS ASN ILE PRO CYS SER ALA LEU LEU SER SER ASP ILE THR ALA SER VAL ASN CYS ALA LYS LYS ILE VAL SER ASP GLY ASN GLY MET ASN ALA TRP VAL ALA TRP ARG ASN ARG CYS LYS GLY THR ASP VAL GLN ALA TRP ILE ARG GLY CYS ARG LEU! In our denaturing medium, at 280 nm εtrp = 5690 M -1 cm -1 and εtyr = 1280 M -1 cm J Biol Chem Aug;238:
16 The experiment! We will determine the concentration of a lysozyme solution indirectly, by first determining the concentration of an aliquot of that solution that we dilute into denaturing conditions. We do that because under denaturing conditions, we can calculate the extinction coefficient because we know the Trp and Tyr content. This extinction coefficient enables us to determine the concentration.! From the dilution factor we will calculate the concentration of the parent native solution.! The calculated concentration and the measured absorbance at 280 nm will then be used to calculate the native protein s extinction coefficient at nm. 32 How we will do it
17 33 Accuracy and precision (Day 1) What you have to do: 1. Use your pipettors to deliver 333 µl, 48µl, and 3µl onto a weighting paper, and report the weight of each drop. 2. Make up a series of standard sample dilutions. 3. Measure absorbance of each. 4. Make a set of identical dilutions. 5. Measure absorbance of each. Step 1a. Use the pipettors in your locker and deionized water from the appropriate faucet near your desk (put some in a beaker). 1b. Go to one of the analytical balances, tare an empty boat then deliver 333 µl water. Record the weight. Repeat for 48 and 3 µl. Accuracy (vs. concentration) Steps 2-3! Your T.A. will provide a concentrated stock solution of Bromophenol blue.! Dilute with water to make the following series of derivative concentrations: 5%, 10%, 20%, 30%, 40%, 60%, 80% (all vol./ vol.).! Measure the absorbance at 590 nm of a cuvette containing only the water used as a diluant.! Rinse the cuvette once with 5% solution and then measure the A590 of this solution.! Repeat for the 10% solution, then the 20%... etc to the highest concentration.! Why should you read from lowest to highest concentration?! Why should you rinse once with solution? 34
18 Accuracy (vs. concentration) Precision! Plot A590 vs. concentration.! Identify the linear regime.! Use the known concentration of the 100% solution to calculate the extinction coefficient based on the linear portion of the plot.! Use the absorbance measured for the 100% solution and the extinction coefficient to calculate the apparent concentration.! How does your apparent concentration compare with the actual concentration?! If you are making a direct determination of concentration and measure an absorbance above 1, what will you do to obtain an accurate value for concentration? Steps 4-5! Make 5 identical 20% solutions by making 5 independent dilutions of 100% solution. In all cases the final volume should be 3 ml.! Measure A590 of each of your solutions and tabulate the results.! Calculate the average value and the standard deviation for your set of readings.! Comment on the results
19 Day 2, Experiment 1: Precautions for Chromogenic assays! The reaction must be limited ONLY by the compound to be measured. (Every molecule of compound is counted.)! A linear relationship must be demonstrated for the absorbance and the reactant that forms the dye.! Conduct the experiment in such a way that the readings corresponding to unknown samples fall within the reading that make up the standard curve.! If necessary, make dilutions of the unknown. Do this BEFORE conducting the reaction. Day 2: juggling two activities. You will be doing two things today: a chromogenic assay and a direct determination. To use your time well you may want to interleave steps of the two activities. A flow chart helps to keep your mind organized as you hop back and forth. Example: Experiment 1 Make series of standard samples Make series of unknown samples Experiment 2 Measure A280 of lysozyme Add dye reagent. Start timing. Add dye reagent. Start timing. Dilute lysozyme w. GuHCl Read and record absorbances. Read and record absorbances. Measure A280 of lysozyme- GuHCl Calculate concentration of initial lysozyme Calculate concentration of diluted lysozyme-guhcl
20 Chromogenic assay What you have to do: 1. Make up standard sample dilutions as per table. 2. Make up unknown sample dilutions as per table. 3. Add 3 ml Bradford reagent to each. Start timer. 4. Gather cuvettes and get in line for spectrophotometer. 5. Once 5 minutes have elapsed read and tabulate A595. (Note how much time had elapsed.) 6. Do not leave without learning from your T.A. what the concentration of the standard is. Chromogenic assay: How to do it The assay conditions! The Bradford concentrate contains methanol and phosphoric acid.! These are potentially hazardous.! How might this formulation be changed for reduced danger?! How will you handle it?! How will you dispose of your reactions?! Standard has a concentration of 2 x 10-2 mm. (Check with your T.A.) 39 40
21 Making standard samples -1 Making standard samples -2! The active ingredient is Coomassie blue G-250, which binds primarily to arginine residues.! The Sigma reagent is used as follows: 3.0 ml reagent ml solution to be tested.! First generate a series of known concentrations of the standard protein solution as follows: Vol. of Protein stdd. soln. (µl) Vol. of buffer (µl)! In your notebook, be sure that you have a name for each sample included in your table and that this name is used to label the corresponding tube.! Make these samples up in test tubes that have sufficient capacity for the addition of 3 ml of the Bradford reagent and subsequent mixing without spilling.! Add 3 ml Bradford reagent to each tube. DO NOT dip a pipette tip into the master bottle, pour a little reagent out of the bottle into a small beaker and pipet from this aliquot. If there is left-over and it is not contaminated, pass it along to a colleague for use
22 Reading the standard samples! Mix each sample and then wait at least 5 min. but no longer than 45 min. (Observe changes in your samples as a function of time, and take photographs.)! Read A595 and record these results. Making the unknown samples! Adapt the protocol detailed above to make a series of five samples of the unknown, plus a null sample.! Your notebook should include a sample table for the unknown that resembles the sample table you made for the standards.! React the unknowns with diluted Bradford reagent as above and read them as above
23 Tips for success Be sure to mix all component thoroughly before taking absorbance measurements The plots of absorbance vs. concentration in the second half of the experiment might not be linear. If so, identify the portion of the plot you will use as the basis for any analysis and explain why. If the color of your unknown tubes is more intense than the known tube with 100 µl of standard, tell the instructor so that the unknown can be remade. Questions! Why do we test a whole series of standard samples instead of just one?! Why do we include a null sample?! Why do we test a whole series of dilutions of our unknown sample instead of just one?! What is a potential pitfall of making a standard curve from one species of protein as a basis for determining the concentration of a different protein?! What are potential hazards of working with the Bradford reagent and why? 46
24 Day 2, Experiment 2: Direct determination of protein concentration! We will determine the concentration of a lysozyme solution under denaturing conditions, because under denaturing conditions, we can calculate the extinction coefficient because we know the Trp and Tyr content. This extinction coefficient enables us to determine the concentration.! From the dilution factor we will calculate the concentration of the parent native solution.! The calculated concentration and the measured absorbance at 280 nm will then be used to calculate the native protein s extinction coefficient ε at 280 nm. Once ε is known the concentration of that protein can be determined without prior denaturation. Direct determination of concentration. What you have to do: 1. Measure A280 of native lysozyme solution using a quartz cuvette. 2. Mix 2.5 ml of the lysozyme with 7.5 ml of guanidinium HCl 3. Rinse cuvette with 3 ml of lysozyme/ GuHCl solution (discard solution used for this). 4. Measure A280 of 3 ml lysozyme/guhcl solution. 5. Measure A280 of another 3 ml lysozyme/guhcl solution
25 Direct determination Protocol: how to do it. (more detail on the steps listed above)! Measure the A280 of a solution of native (folded) lysozyme provided by your T.A. (This will be in 20 mm phosphate buffer at ph 6.5). Use a quartz cuvette for this measurement.! Recover the native lysozyme from the cuvette. Dilute 2.5 ml of the native lysozyme with 7.5 ml of 8 M guanidinium hydrochloride in the same phosphate buffer.! What will be the final concentration of guanidinium hydrochloride?! By what factor will the denatured lysozyme concentration be related to the original concentration of native lysozyme? 49 Questions to be answered in your theory section and pre-lab.! What is the extinction coefficient of denatured lysozyme at 280 nm in our denaturing medium? (Show your work.)! What potential hazards are associated with working with guanidinium HCl?! Does this material contain HCl?! Look at the MSDS for guanidinium HCl available on the course web site and assess this material s toxicity by comparing its LD50 with those of glucose and aspiring, which are also posted.! Why must we use a quartz cuvette? 50
26 Protocol, continued! Transfer 3 ml of denatured lysozyme to the cuvette you just used and then discard this solution. (This step washes away residues of the previous sample.)! Transfer a second 3 ml aliquot of denatured lysozyme to the cuvette and measure its A280.! Discard this solution too and transfer a third 3 ml aliquot of denatured lysozyme to the cuvette and measure its A280 (second measurement). Questions! How do your two readings for the A280 of denatured lysozyme compare?! How does it help to have two readings?! What value should you use and how will you know if it is valid?! Use your calculated extinction coefficient for denatured lysozyme to determine the concentration of denatured lysozyme.! Now calculate the lysozyme concentration of the original native solution of lysozyme based on the dilution factor used.! Use this and the A280 you measured for native lysozyme to calculate the extinction coefficient of native lysozyme
27 Post-lab thoughts! How does the extinction coefficient you determined for native lysozyme compare with published values? (look up at least one, and provide your source. Remember that lysozyme from different sources can have different extinction coefficients, you are looking for hen egg white lysozyme. Hen is also known as Galus galus.! Compare your measured absorbances for the unknown with those measured by a classmate (say who). Discuss the magnitude and possibles causes of any differences.! Also answer questions 2, 3, 4 from our text, experiment 1 (page 24). 53
Background BIOCHEMISTRY LAB CHE-554. Experiment #1 Spectrophotometry
BIOCHEMISTRY LAB Experiment #1 Spectrophotometry CHE-554 In day 1 we will use spectrophotometry as an analytical technique using a known extinction coefficient to assess the precision and accuracy of common
More informationCHMI 2227 EL. Biochemistry I. Test January Prof : Eric R. Gauthier, Ph.D.
CHMI 2227 EL Biochemistry I Test 1 26 January 2007 Prof : Eric R. Gauthier, Ph.D. Guidelines: 1) Duration: 55 min 2) 14 questions, on 7 pages. For 70 marks (5 marks per question). Worth 15 % of the final
More informationExperiment#1 Beer s Law: Absorption Spectroscopy of Cobalt(II)
: Absorption Spectroscopy of Cobalt(II) OBJECTIVES In successfully completing this lab you will: prepare a stock solution using a volumetric flask; use a UV/Visible spectrometer to measure an absorption
More informationSkill Building Activity 2 Determining the Concentration of a Species using a Vernier Spectrometer
Skill Building Activity 2 Determining the Concentration of a Species using a Vernier Spectrometer Purpose To use spectroscopy to prepare a Beer s Law plot of known dilutions of copper(ii) sulfate so that
More informationExperiment 18 - Absorption Spectroscopy and Beer s Law: Analysis of Cu 2+
Experiment 18 - Absorption Spectroscopy and Beer s Law: Analysis of Cu 2+ Many substances absorb light. When light is absorbed, electrons in the ground state are excited to higher energy levels. Colored
More informationExperiment 2: The Beer-Lambert Law for Thiocyanatoiron (III)
Chem 1B Saddleback College Dr. White 1 Experiment 2: The Beer-Lambert Law for Thiocyanatoiron (III) Objectives To use spectroscopy to relate the absorbance of a colored solution to its concentration. To
More informationExperiment 2: The Beer-Lambert Law for Thiocyanatoiron (III)
Chem 1B Dr. White 11 Experiment 2: The Beer-Lambert Law for Thiocyanatoiron (III) Objectives To use spectroscopy to relate the absorbance of a colored solution to its concentration. To prepare a Beer s
More informationExploration of Protein Folding
Exploration of Protein Folding Question: What conditions affect the folding of a protein? Pre-lab reading Atkins & Jones (5 th ed.): Sections 5.1 5.5; 9.8 9.9; and Section 19.13 Safety and Waste Disposal
More informationProtein assay. Absorbance Fluorescence Emission Colorimetric detection BIO/MDT 325. Absorbance
Protein assay Absorbance Fluorescence Emission Colorimetric detection BIO/MDT 325 Absorbance Using A280 to Determine Protein Concentration Determination of protein concentration by measuring absorbance
More informationLecture 5. More on UV-visible Spectrophotometry: Beer s Law and Measuring Protein Concentration
Biological Chemistry Laboratory Biology 3515/Chemistry 3515 Spring 2018 Lecture 5 More on UV-visible Spectrophotometry: Beer s Law and Measuring Protein Concentration 23 January 2018 c David P. Goldenberg
More informationChemistry 118 Laboratory University of Massachusetts Boston Beer s Law
Name: LEARNING GOALS: Chemistry 118 Laboratory University of Massachusetts Boston Beer s Law 1. Become familiar with the concept of concentration and molarity. 2. Become familiar with making dilutions
More informationChem 321 Lecture 18 - Spectrophotometry 10/31/13
Student Learning Objectives Chem 321 Lecture 18 - Spectrophotometry 10/31/13 In the lab you will use spectrophotometric techniques to determine the amount of iron, calcium and magnesium in unknowns. Although
More informationFluorescence Workshop UMN Physics June 8-10, 2006 Basic Spectroscopic Principles Joachim Mueller
Fluorescence Workshop UMN Physics June 8-10, 2006 Basic Spectroscopic Principles Joachim Mueller Fluorescence, Light, Absorption, Jablonski Diagram, and Beer-Law First stab at a definition: What is fluorescence?
More informationProtein Quantitation using a UV-visible Spectrophotometer
UV-0003 UV-visible Spectrophotometer Introduction Generally, protein quantitation can be made using a simple UV-Visible spectrophotometer. The V-630 Bio (Figure 1) is a UV- Visible spectrophotometer designed
More informationProtein assay of SpectroArt 200
Technical Bulletin 14 SpectroArt 200 12/01/2008 Protein assay of SpectroArt 200 MATERIAL BSA: Albumin, bovine serum (Sigma) PBS: BupH TM Phosphate Buffered Saline packs (PIERCE) Bradford assay: Bio-Rad
More informationModule 3. Establishing Standard Curve (Bradford Assay)
Module 3. Establishing Standard Curve (Bradford Assay) 1. Melissa Myoglobin and the Missing Molecules Melissa Myoglobin had a little tube filled with a clear protein solution. To do more research with
More informationA. Two of the common amino acids are analyzed. Amino acid X and amino acid Y both have an isoionic point in the range of
Questions with Answers- Amino Acids & Peptides A. Two of the common amino acids are analyzed. Amino acid X and amino acid Y both have an isoionic point in the range of 5.0-6.5 (Questions 1-4) 1. Which
More informationColorimetry Extinction coefficient ( ) Lambda max ( max) Qualitative vs. quantitative analysis
Lab Week 2/3 - Spectrophotometry Purpose: Introduce students to the use of spectrophotometry for qualitative (what is it) and quantitative (how much is there of it) analysis of biological samples and molecules.
More informationCHEM 254 EXPERIMENT 9. Chemical Equilibrium-Colorimetric determination of equilibrium constant of a weak acid
CHEM 254 EXPERIMENT 9 Chemical Equilibrium-Colorimetric determination of equilibrium constant of a weak acid For a weak acid that can only partly dissociate the equilibrium constant is related to activities
More informationRate law Determination of the Crystal Violet Reaction Using the Isolation Method
Rate law Determination of the Crystal Violet Reaction Using the Isolation Method Introduction A common challenge in chemical kinetics is to determine the rate law for a reaction with multiple reactants.
More informationSpectrometric Determination of the Acid Dissociation Constant of an Acid-base Indicator
Spectrometric Determination of the Acid Dissociation Constant of an Acid-base Indicator Learning Goals 1. Gain appreciation of the dynamics of perturbing a chemical equilibrium 2. Gain an understanding
More informationSo, What Does it Indicate?
So, What Does it Indicate? Introduction Phenolphthalein is a common indicator you may have used in a previous science course, such as Chemistry 130 or Chemistry 170. In solutions with a ph of less then
More informationProteins: Characteristics and Properties of Amino Acids
SBI4U:Biochemistry Macromolecules Eachaminoacidhasatleastoneamineandoneacidfunctionalgroupasthe nameimplies.thedifferentpropertiesresultfromvariationsinthestructuresof differentrgroups.thergroupisoftenreferredtoastheaminoacidsidechain.
More informationEXPERIMENT 14. ACID DISSOCIATION CONSTANT OF METHYL RED 1
EXPERIMET 14. ACID DISSOCIATIO COSTAT OF METHYL RED 1 The acid dissociation constant, Ka, of a dye is determined using spectrophotometry. Introduction In aqueous solution, methyl red is a zwitterion and
More informationOther Methods for Generating Ions 1. MALDI matrix assisted laser desorption ionization MS 2. Spray ionization techniques 3. Fast atom bombardment 4.
Other Methods for Generating Ions 1. MALDI matrix assisted laser desorption ionization MS 2. Spray ionization techniques 3. Fast atom bombardment 4. Field Desorption 5. MS MS techniques Matrix assisted
More informationQuantitative Determination of Proteins
Application Note UV-0003-E Quantitative Determination of Proteins Generally, the concentration of proteins is measured using UV-Vis spectrophotometers. The JASCO V-630 Bio (Figure 1) is a UV/Vis spectrophotometer
More informationExperiment 13I THE REACTION OF RED FOOD COLOR WITH BLEACH 1
Experiment 13I FV 1/11/16 THE REACTION OF RED FOOD COLOR WITH BLEACH 1 PROBLEM: Determine the rate law for the chemical reaction between FD&C Red Dye #3 and sodium hypochlorite. LEARNING OBJECTIVES: By
More informationExperiment 13H THE REACTION OF RED FOOD COLOR WITH BLEACH 1
Experiment 13H 08/03/2017 AHRM THE REACTION OF RED FOOD COLOR WITH BLEACH 1 PROBLEM: Determine the rate law for the chemical reaction between FD&C Red Dye #3 and sodium hypochlorite. LEARNING OBJECTIVES:
More informationKang, Lin-Woo, Ph.D. Professor Department of Biological Sciences Konkuk University Seoul, Korea nd Semester
Kang, Lin-Woo, Ph.D. Professor Department of Biological Sciences Konkuk University Seoul, Korea 2018. 2 nd Semester Absorbance Assay (280 nm) Considerations for use Absorbance assays are fast and
More informationNH 2. Biochemistry I, Fall Term Sept 9, Lecture 5: Amino Acids & Peptides Assigned reading in Campbell: Chapter
Biochemistry I, Fall Term Sept 9, 2005 Lecture 5: Amino Acids & Peptides Assigned reading in Campbell: Chapter 3.1-3.4. Key Terms: ptical Activity, Chirality Peptide bond Condensation reaction ydrolysis
More informationExam I Answer Key: Summer 2006, Semester C
1. Which of the following tripeptides would migrate most rapidly towards the negative electrode if electrophoresis is carried out at ph 3.0? a. gly-gly-gly b. glu-glu-asp c. lys-glu-lys d. val-asn-lys
More informationDETERMINATION OF K c FOR AN EQUILIBRIUM SYSTEM
DETERMINATION OF K c FOR AN EQUILIBRIUM SYSTEM 1 Purpose: To determine the equilibrium constant K c for an equilibrium system using spectrophotometry to measure the concentration of a colored complex ion.
More informationExperiment 7 Buffer Capacity & Buffer Preparation
Chem 1B Dr. White 57 Experiment 7 Buffer Capacity & Buffer Preparation Objectives To learn how to choose a suitable conjugate acid- base pair for making a buffer of a given ph To gain experience in using
More informationMOLEBIO LAB #4: Using a Spectrophotometer
Introduction: Spectrophotometry MOLEBIO LAB #4: Using a Spectrophotometer Many kinds of molecules interact with or absorb specific types of radiant energy in a predictable fashion. For example, when while
More informationChem 460 Laboratory Fall 2008 Experiment 3: Investigating Fumarase: ph Profile, Stereospecificity and Thermodynamics of Reaction
1 Chem 460 Laboratory Fall 2008 Experiment 3: Investigating Fumarase: ph Profile, Stereospecificity and Thermodynamics of Reaction Before Lab Week 1 -- ph Profile for Fumarase Read Box 11-1 (page 323)
More informationEXPERIMENT #3 A Beer's Law Study
OBJECTVES: EXPERMENT #3 A Beer's Law Study To operate a Spectronic 20 To convert from percent transmission to absorbance units To plot absorbance versus wavelength and find max To plot absorbance versus
More informationSo, What Does it Indicate?
So, What Does it Indicate? Introduction Phenolphthalein is a common indicator you may have used in a previous science course, such as Chemistry 184. In solutions with a ph of less then 8.3, this compound
More informationBio 120 Lab 5: Quantitative Analysis
Bio 120 Lab 5: Quantitative Analysis Remember from the measurement lab, concentration is the amount of a solute, also called an analyte, in a given amount of solution. We need a way to measure the concentration
More informationSpectroscopy. Page 1 of 8 L.Pillay (2012)
Spectroscopy Electromagnetic radiation is widely used in analytical chemistry. The identification and quantification of samples using electromagnetic radiation (light) is called spectroscopy. Light has
More informationExperiment 10 Dye Concentration Using a UV-Vis Spectrophotometer
Experiment 10 Dye Concentration Using a UV-Vis Spectrophotometer version 2 Lynta Thomas, Ph.D. and Laura B. Sessions, Ph.D. In this experiment, you will determine the concentration of Allura Red Dye (FD&C
More informationSpectroscopy of Atoms and Molecules
CHEM 121L General Chemistry Laboratory Revision 2.1 Spectroscopy of Atoms and Molecules Learn about the Interaction of Photons with Atoms and Molecules. Learn about the Electronic Structure of Atoms. Learn
More informationExperiment 7. Determining the Rate Law and Activation Energy for the Reaction of Crystal Violet with Hydroxide Ion
Experiment 7. Determining the Rate Law and Activation Energy for the Reaction of Introduction In this experiment, you will observe the reaction between crystal violet and sodium hydroxide. Crystal violet
More informationExperiment 11 Beer s Law
Experiment 11 Beer s Law OUTCOMES After completing this experiment, the student should be able to: determine the wavelength (color) of maximum absorbance for a solution. examine the relationship between
More informationExperiment 13. Dilutions and Data Handling in a Spreadsheet rev 1/2013
Absorbance Experiment 13 Dilutions and Data Handling in a Spreadsheet rev 1/2013 GOAL: This lab experiment will provide practice in making dilutions using pipets and introduce basic spreadsheet skills
More informationChemistry 141 Laboratory Spectrometric Determination of Iron Concentration Lab Lecture Notes 8/29/2011 Dr. Abrash
Chemistry 141 Laboratory Spectrometric Determination of Iron Concentration Lab Lecture Notes 8/29/2011 Dr. Abrash What is the purpose of this experiment? We re going to learn a way to quantify the amount
More informationConcepts, Techniques. Concepts, Techniques 9/11/2012. & Beer s Law. For a simple, transparent, COLORED material, e.g. ROYGBV
9//22 OBJECTIVES Spectrophotometry of Food Dyes & Beer s Law Last Update: 9//22 9:54 AM What is the quantitative basis for the color of substances? How is the absorption/transmission of light measured?
More informationExp 03 - Reaction Rate
GENERAL CHEMISTRY II CAÑADA COLLEGE SUMMER 2018 Exp 03 - Reaction Rate How the speed at which quantities change during a chemical reaction can be measured, predicted and used to understand the mechanism
More informationSpectroscopy Chapter 13
Spectroscopy Chapter 13 Electromagnetic Spectrum Electromagnetic spectrum in terms of wavelength, frequency and Energy c=λν c= speed of light in a vacuum 3x108 m/s v= frequency in Hertz (Hz s-1 ) λ= wavelength
More informationLab Investigation 4 - How could you make more of this dye?
Lab Investigation 4 - How could you make more of this dye? USING SPECTROSCOPY TO DETERMINE SOLUTION CON- CENTRATION Guiding Question How could you make more of this dye? INTRODUCTION A solution is a homogeneous
More informationDetermination of an Equilibrium Constant
Last updated 1/29/2014 - GES Learning Objectives Students will be able to: Determine the numerical value of an equilibrium constant from measured concentrations of all reaction species. Use an absorption
More information1 st European Union Science Olympiad in Dublin, Ireland. task B
1 st European Union Science Olympiad in Dublin, Ireland task B Task B The Properties of Proteins Introduction In this task you will investigate some of the properties of proteins. Proteins consist of a
More information4.3A: Electronic transitions
Ashley Robison My Preferences Site Tools Popular pages MindTouch User Guide FAQ Sign Out If you like us, please share us on social media. The latest UCD Hyperlibrary newsletter is now complete, check it
More informationExperiment 11 Beer s Law
Experiment 11 Beer s Law OUTCOMES After completing this experiment, the student should be able to: determine the wavelength (color) of maximum absorbance for a solution. examine the relationship between
More informationwe might also expect the reaction rate to be influenced by ph. In fact, the rate has been reported to follow the rate law:
KINETICS Objective: The objective of this lab is to measure the rate of iron oxidation, to determine the order of the reaction, and thereby to gain familiarity with rate laws in both the differential and
More informationProtein quantification and detection methods
Protein quantification and detection methods 1) Spectroscopic procedures 2) Measurement of the total protein content by colorimetry 3) Amino acid analysis 4) Other methods, eg. radiolabelling of proteins,
More informationCHEMICAL KINETICS E + 2B 2C + D (1)
CHEMICAL KINETICS Chemical kinetics is the branch of chemistry that is concerned with the study of the rates and mechanisms of chemical reactions. The rate of a reaction is a measure of its speed. Consider
More informationFor simplicity, we ll represent BTB s ionization in a solution by the equilibrium: HBTB = H + + BTB -
Chemistry 160 Please have the following pages ready before class on Wednesday, April 11. An abstract (see the end of this handout) is needed for this write-up. The abstract and photocopied pages of the
More informationLAB #1: ABSORPTION SPECTRA OF CONJUGATED DYES
Chemistry 7 Gustavus Adolphus College LAB #1: ABSORPTIO SPECTRA OF COJUGATED DYES Abstract Ultraviolet-visible spectroscopy is used to explore the electronic structure of several conjugated polyene dyes,
More informationPotentiometric Titration of an Amino Acid. Introduction
NAME: Course: DATE Sign-Off: Performed: Potentiometric Titration of an Amino Acid Introduction In previous course-work, you explored the potentiometric titration of a weak acid (HOAc). In this experiment,
More informationPaper: 12, Organic Spectroscopy Module: 5, Applications of UV spectroscopy
Subject Chemistry Paper No and Title Module No and Title Module Tag Paper 12: Organic Spectroscopy Applications of UV-visible Spectroscopy CHE_P12_M5 TABLE OF CONTENTS 1. Learning Outcomes 2. Introduction
More informationViewing and Analyzing Proteins, Ligands and their Complexes 2
2 Viewing and Analyzing Proteins, Ligands and their Complexes 2 Overview Viewing the accessible surface Analyzing the properties of proteins containing thousands of atoms is best accomplished by representing
More informationAtomic Theory: Spectroscopy and Flame Tests
Atomic Theory: Spectroscopy and Flame Tests Introduction Light energy is also known as electromagnetic (EM) radiation. The light that we observe with our eyes, visible light, is just a small portion of
More informationExperiment 3. Condensation Reactions of Ketones and Aldehydes: The Aldol Condensation Reaction.
Experiment 3. Condensation Reactions of Ketones and Aldehydes: The Aldol Condensation Reaction. References: Brown & Foote, Chapters 16, 19, 23 INTRODUCTION: This experiment continues the saga of carbon-carbon
More informationSpectrophotometric Determination of an Equilibrium Constant
Spectrophotometric Determination of an Equilibrium Constant v021214 Objective To determine the equilibrium constant (K c ) for the reaction of iron (III) ion with thiocyanate (SCN - ) to form the thiocyanatoiron(iii)
More informationGE Healthcare Life Sciences. Spectrophotometry. Handbook
GE Healthcare Life Sciences Spectrophotometry Handbook Contents Spectrophotometry basics 3 What is spectrophotometry? 3 Definition 3 Lambert s Law 4 Beer s Law 4 Nucleic acid applications 6 Direct UV measurement
More informationChem 2115 Experiment #10. Acids, Bases, Salts, and Buffers
Chem 2115 Experiment #10 Acids, Bases, Salts, and Buffers OBJECTIVE: The goal of this series of experiments is to investigate the characteristics of acidic and basic solutions. We will explore the neutralization
More informationLaboratory Measurements and Procedures
18 Introduction Measurements of masses, volumes, and preparation of chemical solutions of known composition are essential laboratory skills. The goal of this exercise is to gain familiarity with these
More informationSpectrophotometry Materials
Spectrophotometry Materials Item per Class per Bench Genesys 10UV Spectrophotometer 6 1 13 ml test tubes box 7 Test tube racks 6 1 1% Albumin solution 25 ml/one flask 2 ml 0.7% Albumin solution (unknown
More informationKinetics of Crystal Violet Fading AP* Chemistry Big Idea 4, Investigation 11 An Advanced Inquiry Lab
Introduction Kinetics of Crystal Violet Fading AP* Chemistry Big Idea 4, Investigation 11 An Advanced Inquiry Lab Catalog o. AP7644S Publication o. 7644S Crystal violet is a common, beautiful purple dye.
More informationEnergy. Position, x 0 L. Spectroscopy and the Particle-in-a-Box. Introduction
Spectroscopy and the Particle-in-a-Box Introduction The majority of colors that we see result from transitions between electronic states that occur as a result of selective photon absorption. For a molecule
More informationCHEMISTRY 135 General Chemistry II. Determination of an Equilibrium Constant
CHEMISTRY 135 General Chemistry II Determination of an Equilibrium Constant Show above is a laboratory sample from chemistry, not phlebotomy. [1] Is the bloody-looking product the main component of this
More informationContent : Properties of amino acids.. Separation and Analysis of Amino Acids
قسم الكيمياء الحيوية.دولت على سالمه د. استاذ الكيمياء الحيوية ٢٠١٥-٢٠١٤ المحاضرة الثانية 1 Content : Properties of amino acids.. Separation and Analysis of Amino Acids 2 3 Physical Properties of Amino
More informationKinetics of Crystal Violet Fading AP Chemistry Big Idea 4, Investigation 11 An Advanced Inquiry Lab (adapted by Flinn Scientific, Inc.
Introduction Kinetics of Crystal Violet Fading AP Chemistry Big Idea 4, Investigation 11 An Advanced Inquiry Lab (adapted by Flinn Scientific, Inc.) Crystal violet is a common, beautiful purple dye. In
More informationExperiment 1 Chemical Kinetics 1
Experiment 1 Chemical Kinetics 1 Purpose: Determine the rate law for the reaction of the dye crystal violet with hydroxide. Reading: Brown, et. al., Chemistry The Central Science, sections 14.1 14.4. Introduction
More informationChemical Kinetics: Determining Rate Laws for Chemical Reactions
Chemical Kinetics: Determining Rate Laws for Chemical Reactions v010816 INTRODUCTION It is thought that the birth of chemical kinetics occurred in 1850 when a German chemist, Ludwig Ferdinand Wilhelny,
More informationExperiment #7. Determination of an Equilibrium Constant
Experiment #7. Determination of an Equilibrium Constant Introduction It is frequently assumed that reactions go to completion, that all of the reactants are converted into products. Most chemical reactions
More informationExperiment 6: Determination of the Equilibrium Constant for Iron Thiocyanate Complex
Experiment 6: Determination of the Equilibrium Constant for Iron Thiocyanate Complex The data for this lab will be taken as a class to get one data set for the entire class. I. Introduction A. The Spectrophotometer
More informationBradford Reagent, 5x
INSTRUCTION MANUAL Bradford Reagent, 5x Reagent for protein quantification (Cat. No. 39222) SERVA Electrophoresis GmbH Carl-Benz-Str. 7 D-69115 HeidelbergPhone +49-6221-138400, Fax +49-6221-1384010 e-mail:
More informationTechniques in Molecular Genetics Spectroscopy and Enzyme Assays
Techniques in Molecular Genetics Spectroscopy and Enzyme Assays H.E. Schellhorn Spectroscopy Chromophore Molar Extinction Coefficient Absorbance Transmittance Spectroscopy Many biological materials have
More informationLAB 05 Enzyme Action
LAB 05 Enzyme Action Objectives: Name the substrate and products of the peroxidase-catalyzed reaction. To understand the terms: enzyme, activation energy, active site, ph, and denaturation. Distinguish
More informationBeer's Law and Data Analysis *
OpenStax-CNX module: m15131 1 Beer's Law and Data Analysis * Mary McHale This work is produced by OpenStax-CNX and licensed under the Creative Commons Attribution License 2.0 1 Beer's Law and Data Analysis
More informationAtomic Theory: Spectroscopy and Flame Tests
Atomic Theory: Spectroscopy and Flame Tests Introduction Light energy is also known as electromagnetic (EM) radiation. The light that we observe with our eyes, visible light, is just a small portion of
More informationANALYSIS OF ZINC IN HAIR USING FLAME ATOMIC ABSORPTION SPECTROSCOPY
ANALYSIS OF ZINC IN HAIR USING FLAME ATOMIC ABSORPTION SPECTROSCOPY Introduction The purpose of this experiment is to determine the concentration of zinc in a sample of hair. You will use both the calibration
More informationExperiment 8: DETERMINATION OF AN EQUILIBRIUM CONSTANT
Experiment 8: DETERMINATION OF AN EQUILIBRIUM CONSTANT Purpose: The equilibrium constant for the formation of iron(iii) thiocyanate complex ion is to be determined. Introduction: In the previous week,
More informationStudent Manual. Background STUDENT MANUAL BACKGROUND. Enzymes
Background Enzymes Enzymes are typically proteins (some nucleic acids have also been found to be enzymes) that act as catalysts, speeding up chemical reactions that would take far too long to occur on
More informationProtein Quantification and. Detection methods. copy the document as long as you keep it intact. It is not allowed to use
Protein Quantification and Detection methods Disclaimer This document is compiled from the publications mentioned above and my own research. The document is provided as is in a good scientific manner to
More informationProtein Quantification Kit (Bradford Assay)
Protein Quantification Kit (Bradford Assay) Booklet Item NO. KTD3002 Product Name Protein Quantification Kit (Bradford Assay) ATTENTION For laboratory research use only. Not for clinical or diagnostic
More informationChemistry 1215 Experiment #11 Spectrophotometric Analysis of an Unknown Brass Sample
Chemistry 1215 Experiment #11 Spectrophotometric Analysis of an Unknown Brass Sample Objective In this experiment you will use spectrophotometric measurements to determine the copper concentration of a
More informationTo observe flame test colors produced by ions in solution.
Flame Tests PURPOSE To determine the identities of ions in two solutions of unknown composition by comparing the colors they produce in flame tests with colors produced by solutions of known composition.
More informationExperiment. Quantification of Ascorbic acid by Fluorescence Spectroscopy1
Experiment. Quantification of Ascorbic acid by Fluorescence Spectroscopy Modified 10/2017 Experiment. Quantification of Ascorbic acid by Fluorescence Spectroscopy1 Objective: The goal of this experiment
More informationColorimetric analysis of aspirin content in a commercial tablet
Colorimetric analysis of aspirin content in a commercial tablet v010214 Objective In this lab, you will prepare standard solutions, and use Beer s Law to construct a calibration curve. You will determine
More informationRedox Titration of Ferricyanide to Ferrocyanide with Ascorbic Acid Spectroelectrochemistry: Redox Titration Using Vitamin C
Redox Titration of Ferricyanide to Ferrocyanide with Ascorbic Acid Spectroelectrochemistry: Redox Titration Using Vitamin C While spectroscopy is often used to directly quantify compounds in solution it
More informationFirst week Experiment No.1 / /2013. Spectrophotometry. 1. Determination of copper via ammine complex formation using standard series method
First week Experiment No.1 / /2013 Spectrophotometry 1. Determination of copper via ammine complex formation using standard series method Principal In the standard series method the teat and standard solution
More informationExperiment 12: SPECTROSCOPY: EMISSION & ABSORPTION
Sample Experiment 12: SPECTROSCOPY: EMISSION & ABSORPTION Purpose: Emission and absorption spectroscopy is to be explored from different perspectives in a multipart experiment. Part I: Certain elements
More informationAtomic Theory: Spectroscopy and Flame Tests
Atomic Theory: Spectroscopy and Flame Tests Introduction Light energy is also known as electromagnetic (EM) radiation. The light that we observe with our eyes, visible light, is just a small portion of
More informationMonensin ELISA Kit. Catalog Number KA assays Version: 11. Intended for research use only.
Monensin ELISA Kit Catalog Number KA1422 96 assays Version: 11 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Background... 3 Principle of the Assay... 3 General Information...
More informationUNIT TWELVE. a, I _,o "' I I I. I I.P. l'o. H-c-c. I ~o I ~ I / H HI oh H...- I II I II 'oh. HO\HO~ I "-oh
UNT TWELVE PROTENS : PEPTDE BONDNG AND POLYPEPTDES 12 CONCEPTS Many proteins are important in biological structure-for example, the keratin of hair, collagen of skin and leather, and fibroin of silk. Other
More informationINSTRUCTION MANUAL. Lowry Assay Kit. Kit for Protein Quantification. (Cat. No )
INSTRUCTION MANUAL Lowry Assay Kit Kit for Protein Quantification (Cat. No. 39236) SERVA Electrophoresis GmbH Carl-Benz-Str. 7 D-69115 Heidelberg Phone +49-6221-138400, Fax +49-6221-1384010 e-mail: info@serva.de
More informationCHAPTER - 3 ANALYTICAL PROFILE. 3.1 Estimation of Drug in Pharmaceutical Formulation Estimation of Drugs
CHAPTER - 3 ANALYTICAL PROFILE 3.1 Estimation of Drug in Pharmaceutical Formulation 3.1.1 Estimation of Drugs ANALYTICAL PROFILE 84 3.1 ESTIMATION OF DRUG IN PHARMACEUTICAL FORMULATION. Agrawal A et al
More informationAIM To verify Beer - Lambert s law and to determine the dissociation constant (Ka) of methyl red, Spectrophotometrically.
C 141(Expt. No. ) NAME : ROLL No. : SIGNATURE : BATCH : DATE : VERIFICATION OF BEER - LAMBERT S LAW & DETERMINATION OF DISSOCIATION CONSTANT (Ka) OF METHYLRED, SPECTROPHOTOMETRICALLY AIM To verify Beer
More information