1. Absorbance Part A Determination of Lambda Max 2. Maximum Absorbance Learning objectives: Determination of the wavelength of maximum absorbance of a given solution Learn how to use spectrophotometer Calculate absorbance 3. Spectrophotometry is a technique used to measure how much energy a substance absorbs at varying wavelengths of light. Wavelength (nm) Absorbance 0.0 2.0 200 250 300 350 400 450 4. What is wavelength? Forms of electromagnetic radiation like radio waves or light waves make characteristic patterns as they travel through space. Each wave has a certain shape and length. The distance between peaks (high points) is called wavelength. 5. UV-Vis spectrophotometry 5 6. UV-Vis Spectrophotometry 6 Color of a solution: 7. Visible light is only a small portion of the entire electromagnetic spectrum it includes the colors commonly observed (red, yellow, green, blue and violet). The visible spectrum consists of electro- magnetic radiation whose wavelengths range from 380nm to nearly 760nm. Background : 8. Wavelength (, Greek lambda): Distance from one wave peak to the next. Units: m, cm, m, nm or A Light waves can be characterized By: 9. Spectrophotometer To measure the amount of light that a sample absorbs Visible spectrophotometer: uses light over visible range (400 - 700 nm) of electromagnetic radiation spectrum. 10. Types of spectrophotometers 11. 11 UV spectrum of phenolphthalein 12. UV-visible spectrum The two main properties of an absorbance peak are: 1. Absorption wavelength max 2. Absorption intensity Amax Housecroft and Sharpe, p. 466 13. Identifying a compound by spectrophotometry If a compound absorbs light its absorption spectrum is a unique property of that compound. The molecular structure is responsible for the absorption properties The most common feature of absorbing compounds are conjugated double bonds, often as an aromatic ring 14. Spectrophotometry By measuring the Absorption Spectrum of a substance, it is possible to identify it or at least place it in a particular class of compounds. Max 15. Absorbance vs Wavelength Different compounds absorb best at different wavelengths 16. Transmittance (T) is defined as the fraction of incident light which is transmitted, ie, passes through, a sample. Thus, T = I/Io, Transmittance 17. Defined as the ratio of the intensity of light emerging from the solution (I) to that of incident light entering (Io) Transmittance 18. Transmittance is usually expressed as a percentage: %T = (I/Io) x 100 100% transmittance means no light is absorbed by the solution so that incident light is 100% transmitted Transmittance 19. Absorbance (A), or optical density, is a logarithmic function of T and is expressed as: A = 2 log(%T) Note that absorbance has no units. Absorbance 20. So, for example, at 100% transmittance, A = log 1.0 = 0. At 50% transmittance, A = 2 -log (50) = 0.30. Transmittance and Absorbance 21. Lets recap 22. The spectrophotometer displays: Absorbance -- a number between 0 and Transmittance -- a number between 0 and 100%. Transmittance and Absorbance 23. SPECTROPHOTOMETER READINGS 24. Applications/Use Identify food dyes Protein assays of milk and protein drinks Test rates of photosynthesis Bacterial growth Use enzyme preparations to break down complex sugars Determine unknown concentration of solution Determine equilibrium constant of a reaction involving ions Purity of protein or nucleic acid preps (A260 /A280) 25. Reference: http://abacus.bates.edu/~ganderso/biology/resources/spec20.html#spectrophotometry How to use Spectrophotometer 26. How to use Spectrophotometer Reference: http://abacus.bates.edu/~ganderso/biology/resources/spec20.html#spectrophotometry 27. Reference: http://abacus.bates.edu/~ganderso/biology/resources/spec20.html#spectrophotometry How to use Spectrophotometer Turn on the Spec 20 to warm it up for 10 minutes (left front knob). Set wavelength using the dial on top Step 1: 28. Prepare a BLANK cuvette by adding WATER A BLANK is used to calibrate the Spec 20 so that any absorbance attributable to the solvent and/or glass cuvette can be compensated. By zeroing the Spec 20 to the blank, you will measure only the absorbance due to the substance in question. Reference: http://abacus.bates.edu/~ganderso/biology/resources/spec20.html#spectrophotometry Step 2: 29. With no tube in the holder, adjust the meter needle to read infinite absorbance (= 0% transmittance) using the left front knob (= power switch) Reference: http://abacus.bates.edu/~ganderso/biology/resources/spec20.html#spectrophotometry Step 3: 30. Using a Kimwipe, wipe off/polish the outside of the cuvette. Using a Sharpie, make a small vertical mark at the top of each cuvette for alignment in the sample holder. Reference: http://abacus.bates.edu/~ganderso/biology/resources/spec20.html#spectrophotometry Step 4: 31. Reference: http://abacus.bates.edu/~ganderso/biology/resources/spec20.html#spectrophotometry Step 5: Insert the cuvette to line up with the line on the sample holder. Close the lid. 32. Reference: http://abacus.bates.edu/~ganderso/biology/resources/spec20.html#spectrophotometry Step 6: The spectrophotometer is now calibrated to this BLANK. Using the right front knob, adjust the meter needle to read absorbance = 0.0 (= 100 % transmittance). 33. Remove BLANK and insert cuvette with your sample. Close lid. Read transmittance (upper scale) for your sample. Repeat steps 3-8 for each wavelength Reference: http://abacus.bates.edu/~ganderso/biology/resources/spec20.html#spectrophotometry Step 7: Step 8: Step 9: Measuring Absorbance or Transmittance 34. NOTE: When taking several measurements at the same wavelength over a short time period, you do not need to re-blank for each. If you are taking readings over an extended period or change the wavelength, you must re- zero the instrument., Re-blank the spectrophotometer 35. Summary 1. Turn instrument on 2. Select correct wavelength (400-700 nm) 3. Block light, set Zero (no light = infinity absorption = 0% T) 4. Choose and clean test-tubes 5. Open light, insert Blank (maximum light = no absorption = 100% T) 6. Measure transmittance of samples 7. Repeat from step 2 36. Method: 1) Prepare solution of Basic Fuchsin Dye a) 1 ml of Dye into 9 ml of water (total 10 ml) 2) Determine spectrum: a) Set up spectrophotometer b) Measure %T using lambda (400 700 nm) c) Convert %T to Absorbance 37. Data Analysis Plot your data on a graph Wavelenght is plotted on the X-axis Absorbance is plotted on the Y-axis Put units on each axis Determine the wavelength of maximum absorbance Now you are ready to proceed to part B 38. LABORATORY #4 Part B: Verification of Beer-Lambert Law 39. Beer-Lambert Law Learning objectives: What is Beer-Lambert Law? Determine the relationship between concentration and absorbance of the solution How to calculate dilutions 40. Transmittance (T) Absorbance (A) A = 2 - log10 (%T) Transmittance and Absorbance 41. The Beer-Lambert law describes that Absorbance, A, is directly proportional to concentration (M): Beer-Lambert Law 42. Absorbance increases linearly with concentration Standard curve 43. What is concentration and how we can calculate it 44. Mole: is simply the amount of a substance that contains 6.02 x 1023 particles. Avogadro's Constant: The number of objects in a mole; 6.02 x 1023 Molarity: A unit of concentration equal to the number moles of solute in a 1 liter of solution. VOCABULARY: 45. What is mole? measurement for the amount of substance a mole of any pure substance has a mass in grams exactly equal to that substance's atomic or molecular mass 46. What is mole? atomic mass for C = 12 g 47. What is mole? MW = 12 g/mole 48. The Mole (n) 1 dozen cookies = 12 cookies 1 mole of cookies = 6.02 X 1023 cookies 1 dozen cars = 12 cars 1 mole of cars = 6.02 X 1023 cars 1 dozen Al atoms = 12 Al atoms 1 mole of Al atoms = 6.02 X 1023 atoms Note that the NUMBER is always the same, but the MASS is very different! 49. Suppose we invented a new collection unit called a rapp. One rapp contains 8 objects. 1. How many paper clips in 1 rapp? a) 1 b) 4 c) 8 2. How many oranges in 2.0 rapp? a) 4 b) 8 c) 16 3. How many rapps contain 40 gummy bears? a) 5 b) 10 c) 20 Learning Check 50. 6.02 x 1023 particles 1 mole or 1 mole 6.02 x 1023 particles Note that a particle could be an atom OR a molecule! Avogadros Number as Conversion Factor 51. 1. Number of atoms in 0.500 mole of Al a) 500 Al atoms b) 6.02 x 1023 Al atoms c) 3.01 x 1023 Al atoms 2.Number of moles of S in 1.8 x 1024 S atoms a) 1.0 mole S atoms b) 3.0 mole S atoms c) 1.1 x 1048 mole S atoms Learning Check 52. Molarity Or Concentration 53. Molarity (M) A concentration that expresses the moles of solute in 1 L of solution Molarity (M) = moles of solute (n) 1 liter solution (V) 54. Molarity (M) Molarity (M) = moles of solute 1 liter solution The solvent is the liquid in which something is dissolved in, usually water Solute is a dissolved substance Solution is a type of homogenous mixture in which the solute is distributed uniformly throughout another substance, the solvent 55. Units of Molarity 2.0 M HCl = 2.0 moles HCl 1 L HCl solution 6.0 M HCl = 6.0 moles HCl 1 L HCl solution 56. Molarity Conversion Factors A solution is a 3.0 M NaOH.. Write the molarity in the form of conversion factors. 3.0 moles NaOH and 1 L NaOH soln 1 L NaOH soln 3.0 moles NaOH 57. How to calculate molarity Example 1: What is the molarity of a 5.00 liter solution that was made with 10.0 moles of KBr ? http://www.fordhamprep.org/gcurran/sho/sho/lessons/lesson64.htm 58. What is the molarity of a 5.00 liter solution that was made with 10.0 moles of KBr ? Solution: # of moles of solute Molarity = -------------------------- Liters of solution Given: # of moles of solute = 10.0 moles Liters of solution = 5.00 liters 10.0 moles of KBr Molarity = -------------------------- = 2.00 M 5.00 Liters of solution Answer = 2.00 M http://www.fordhamprep.org/gcurran/sho/sho/lessons/lesson64.htm 59. GOLDEN RULE: ALWAYS TO CHECK UNITS!!! 60. Learning Check M1 A KOH solution with a volume of 400 mL contains 2 mole KOH. What is the molarity of the solution? 1) 0.8 M 2) 5 M 3) 0.005 M Molarity (M) = moles of solute 1 liter solution 61. Solution M1 A KOH solution with a volume of 400 mL contains 2 moles of KOH. What is the molarity of the solution? 2) 5 M M = 2 mole KOH = 5 M 0.4 L 62. Learning Check M3 Stomach acid is a 0.10 M HCl solution. How many moles of HCl are in 1500 mL of stomach acid solution? 1) 15 moles HCl 2) 1.5 moles HCl 3) 0.15 moles HCl 63. Solution M3 3) 1500 mL x 1 L = 1.5 L 1000 mL 1.5 L x 0.10 mole HCl = 0.15 mole HCl 1 L (Molarity factor) 64. What is dilution? The act of diluting a solution is to simply add more water (the solvent) thus leaving the amount of solute unchanged 65. What is dilution? the amount of solute is unchanged = 13 = 13 66. Amount or moles of solute before dilution (nb) and the moles of solute after the dilution (na) are the same: nb = na MOLARITY & DILUTION = 13 = 13 67. MOLARITY & DILUTION MbVb = MaVa Remember that M = So, the moles for any solution can be calculated by n=MV Moles (n) 1 Liter (V) 68. MOLARITY & DILUTION MbVb = MaVa Since n=MV A relationship can be established such that MbVb = nb = na = MaVa Or simply : MbVb = MaVa 69. MOLARITY & DILUTION MbVb = MaVa 70. How to calculate serial dilutions Dilution Equation: MbVb = MaVa Mb = Molarity of initial solution Vb = Volume of initial solution to be used Ma = Molarity of final (diluted) solution Va = Desired volume of final dilute solution. 71. How to calculate serial dilutions Dilution Equation: MbVb = MaVa Mb = Molarity of initial solution Vb = Volume of initial solution to be used Ma = Molarity of final (diluted) solution Va = Desired volume of final dilute solution. 2 ml 2 ml of water 1 M 72. How to calculate serial dilutions Dilution Equation: MbVb = MaVa 2 ml 2 ml of water 1 MMb Vb Va = 2ml +2 ml = 4 ml Ma = ??? MbVb = MaVa Ma = MbVb Va = (1M)x(2ml) 4 ml = 0.5 M 73. How to calculate serial dilutions Dilution Equation: MbVb = MaVa Mb = Molarity of initial solution Vb = Volume of initial solution to be used Ma = Molarity of final (diluted) solution Va = Desired volume of final dilute solution. 2 ml 2 ml of water 1 MMb Vb Va = 2ml +2 ml = 4 ml Ma = ??? 74. GOLDEN RULE: ALWAYS TO CHECK UNITS!!! 75. MOLARITY & DILUTION Calculate the molarity of a solution prepared by diluting 25.0 mL of 0.05 M potassium iodide with 50.0 mL of water. Mb = 0.05 mol/L Ma = ? Vb = 25.0 mL Va = 50.0 + 25.0 = 75.0 mL 76. MOLARITY & DILUTION Calculate the molarity of a solution prepared by diluting 25.0 mL of 0.05 M potassium iodide with 50.0 mL of water. Mb = 0.05 mol/L Ma = ? Vb = 25.0 mL Va = 50.0 + 25.0 = 75.0 mL MbVb = MaVa Mb Vb = Ma = (0.05 mol/L) (25.0 mL) = 0.0167 M of Ka Va 75.0 mL 77. Example: What volume of 0.01 M solution do we need to make 20 mL of a 0.001 M solution? 78. Example: What volume of 0.01 M solution do we need to make 20 mL of a 0.001 M solution? Use the dilution equation, MbVb = MaVa Mb=0.01 M Vb = unknown Ma= 0.001 M Va = 20 mL Solve for Vb 79. Example: What volume of 0.01 M solution do we need to make 20 mL of a 0.001 M solution? Use the dilution equation, MbVb = MaVa Mb=0.01 M Vb = unknown Ma= 0.001 M Va = 20 mL Vb=(0.001M)x(20ml) = ??? 0.01M 80. Example: What volume of 0.01 M solution do we need to make 20 mL of a 0.001 M solution? Got 2 mL. This is the amount of the 0.01 M solution that we need. The total volume is 20 mL, so we need to add 18 mL of water. So, measure 2 mL of your 0.01 M solution and add it to 18 mL of water to make a 0.001 M solution. 81. Methods: 1) Prepare serial dilutions using Basic Fuschin Dye a) Stock (1 ml Dye + 9 ml of water) b) Set up different concentrations of dye 2) Determine %T: a) Set up spectrophotometer b) Measure %T using maximum lambda from Part A c) Convert %T to Absorbance d) Plot Absorbance vs. Concentration 82. Plot Absorbance vs. Concentration 83. How to make dilutions (flow chart) 9 ml Water (mL)4.5 4.0 3.5 3.0 Stock solution: 1 ml dye + 9 ml water 0.5 1.0 1.5 2.0 Stock (mL) 2.5 2.5 ml H2O 1 6 5432 Mb=0.0024M MbVb = MaVa 84. Show your calculations, proper graphs and results Be neat Part A and Part B are due before the class next week (week 5) 85. Just for fun More info for chemistry nerds!!! 86. 87 Concentration Absorbance,A 0 0.5 1 Concentration Transmittance,T A=bc certain constant b One analyte T=10-A =10- bc Beers law is a relation between absorbance and concentration which is a straight line passes by origin at constant pathlength, b, and at certain wavelength, . Transmittance decreases exponentially as concentration increases Beers law is obeyed for monochromatic light 50% 25% 12.5% P0 = 100% Solution with T = 50% not zero Slope = b 87. UV/visible: Applications UV/visible is still used in current research, especially for heme-containing proteins, which have absorbance in the Soret region that is sensitive to the state of the protein 3d shell of Fe2+ has 6 electrons High Spin Low Spin 88. UV/Visible Applications This paper looks at iron-sulfur clusters in a native and a mutant protein C196S Mutant lacks broad absorption band between 400-600 nm which is diagnostic of an 2Fe- 2S cluster JBC (1998)Vol. 273, No. 35;28 pp. 2231122316 89. Applications of Spectrophotometer Spectroscopy Chemical Analysis: concentration ,trace analysis, pH and remote monitoring Geology. Astronomy. Particle size. Thin film characterization Color matching Optics 90. For the mathematically minded: Transmittance = IT/I0*100 Absorbance = log10(I0/IT) Converting Transmittance to Absorbance %T/100 = IT/I0 100/%T = I0/IT Taking logs on both sides Log 100 log %T = log I0/IT 2 log%T = Absorbance