Concepts, Techniques. Concepts, Techniques 9/11/2012. & Beer s Law. For a simple, transparent, COLORED material, e.g. ROYGBV

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1 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? How is the intensity of color related to the concentration of colored solutions? What considerations go into graphing experimental data? Questions 2 Concepts: Absorptivity Absorption Concepts, Techniques Analytical Wavelength Beer s Law Blank Concentration Electromagnetic Spectrum Incident Intensity Spectroscopy Spectrum Transmittance Wavelength Techniques: Concepts, Techniques Visible Spectroscopy Quantitative dilution Graphing Apparatus: Plotting /Curve fitting Recording Spectrophotometer Spectronic 2 Pipet/Syringe Cuvette Purpose 3 Purpose 4 BACKGROUND For a simple, transparent, COLORED material, e.g. ROYGBV ROYGBV Incident white light I i (λ) VIOLET CLEAR SOLUTION Transmitted light I t (λ) Can plot a graph showing the percent of light of each discrete COLOR (wavelength, λ) transmitted by the solution Percent Transmittance I t (λ) %T(λ) = X I i (λ) 5 % Tran TRA nsmitted NSM ITTED % Transmittance of of White Light Light by a Violet by Solution a Violet Solution UV 3 2 Violet Blue Or, instead, we can plot the percent of light absorbed Wavelength WAVELENGTH (nm) (nm) Green Yellow Red IR 6

2 9//22 % Ab bsorbed % Absorbed of White of Light by Violet by a Solution Violet Solution % Absorbed = % - % Transmitted WAVELENGTH (nm) Violet Blue Yellow Red Green 7 What determines the WAVELENGTHS of light that substances absorb? VISIBLE AND ULTRAVIOLET: Electronic structure of molecules INFRARED REGION: Vibrations of molecules What determines INTENSITY of absorption? EFFICIENCY of INTERACTION between Molecules & Electromagnetic Radiation 8 Which DEVICE measures this phenomenon? A SPECTROPHOTOMETER measures the RELATIVE absorption of light of DIFFERENT WAVELENGTHS by a substance SOURCE of light, MONOCHROMATOR an element to select particular wavelengths SAMPLE & CONTAINER Detector output depends on intensity, wavelength & instrumental factors. Other substances may also absorb. Therefore, we always measure RELATIVE output of detector, where RELATIVE means compared with: EVERYTHING BUT THE SAMPLE ( a BLANK ) Sample container Solvent Other reagents, etc. - = ELECTRONICS to translate detector output into numbers DETECTOR to measure intensity of light transmitted through sample, and 9 % % % BLANK + SAMPLE BLANK SAMPLE This insures that measured of light is due only to the substance of interest How is the amount of COLOR absorbed by a chemical species related to the CONCENTRATION of that species? BEER-LAMBERT LAW (76-852) Amount of light absorbed by a transparent solution is proportional to the thickness of the solution & concentration of the absorbing species absorbance = constant x path length x concentration A = ε b c A = ABSORBANCE at a specified wavelength ε = ABSORPTIVITY, a constant characteristic of the substance at that wavelength (does not depend on the amount or concentration of the sample) b = PATH LENGTH of the light through sample, i.e., cell thickness c = CONCENTRATION of the absorbing substance Definition of A = log How is related to %Transmittance? A = log %T I I t Smaller I t means larger A I t %T = I I = log - log %T A = log %T 2 2

3 9//22 Need to know: A = ε b c What do we need to know to relate to concentration? ABSORPTIVITY, ε, AND THICKNESS OF THE CUVETTE, b at least, OR their product, ε b 3 How do we proceed experimentally?.) Determine the substance s SPECTRUM in the desired wavelength range i.e. absorbance as a function of wavelength 2.) Find WAVELENGTH at which h the substance has the LARGEST ABSORBANCE (lowest %Transmittance) The will be most sensitive to concentration at this wavelength vs Wavelength Wavelength (nm) This wavelength is called the ANALYTICAL WAVELENGTH 4 2.) Then we prepare series of solutions of KNOWN CONCENTRATIONS of the substance c/8 c/4 c/2 c 3.) Measure ABSORBANCE of each solution at its ANALYTICAL WAVELENGTH Tabulate the data Conc (mg/l) Abs BLANK and 4.) Plot ABSORBANCE vs CONCENTRATION 5 6 vs Concentration Red Dye 47 nm SUPL-4 Slope of Beer s Law plot is ε X b, where BLANK SLOPE =.658 / 25. =.263 L/mg Concentration (mg / L) Note : is dimensionless. 7 ε = the absorptivity of the substance b = the cell thickness If we use identical (i.e., matched) cells for blank & sample, we do not need to know the quantities ε & b separately. Beer s Law expression for Red #2 at its analytical wavelength in this cell becomes: A =.263 X C (where C is in mg/l) Units of the slope of the Beer s Law plot are (always) reciprocal concentration (e.g., L / mg) 8 3

4 9//22 In this exercise, we examine spectrophotometric properties of 7 FOOD DYES 7 substances approved by Food and Drug Administration ( FDA ), for use as colorants in foods Each dye is a single complex organic compound. For simplicity they are called: Blue, Blue 2, Green 3, Yellow 5, Yellow 6, Red 3, and Red 4 We will further examine dyes and their chemical properties next week 9 PROCEDURE WORK IN PAIRS. Pair obtains 5 ml of ONE assigned dye solution 2. Determine SPECTRUM (abs vs ) of diluted stock solution between 35 nm and 75 nm Using recording spectrophotometer 3. Determine ANALYTICAL WAVELENGTH, i.e., wavelength of maximum absorbance. 4. Prepare dilutions of stock solution of precisely known concentration: 3/4, /2, /4, /8, and /6 5. Determine ABSORBANCE of stock solution & dilutions at analytical wavelength most dilute solution (/6) first, 2 Procedure Schedule. You will be assigned a partner and ONE dye 2. All pairs begin by preparing dilutions for Beer s Law While doing that: TAs send one pair at the time to the recording spectrophotometer to obtain spectrum of the dye. 2 PAIRS will SHARE ONE SPECTRONIC 2 In In part Part 2 2 (Beer s Law Law) - Groups of two should use spectrophotometers in series i.e., group does complete Beer s Law data acquisition sets wavelength This avoids resetting check blank spectrophotometer after measure dye / conc each measurement measure dye / conc 2, etc. When group finishes, group 2 resets Spectronic 2 and does the same for its dye Prepare BEER'S LAW PLOT BY HAND! Use rules in SUPL vs Concentration Red Dye #2 at 47 nm Concentration (mg/l) Concepts - Graphing: Concepts, Techniques Abscissa Ordinate Scale Intercept Dependent Variable Portrait Orientation Graph Slope Independent Variable Grid Landscape Range Grid Lines 23 Purpose 24 4

5 9//22 SUPL-4 Summary The principal rules for drawing graphs described in SUSB-4 are: The abscissa (x-axis) shall generally represent the independent variable. The dependent variable is therefore represented by the ordinate (y-axis) The scales s shall use, 2, or 5 units or decimal submultiples or multiples thereof per mm. I.e., mm =.,.2,.5,.,.2,.5,, 2, 5, units The orientation (portrait or landscape) shall be chosen to maximize the plot area mm marks Portrait or Landscape? Box (mm) must represent a multiple or submultiple of, 2 or 5 units (not 3 or 4) 8 mm: 8, 8/2 = 9, 8/5 = 36, 8/ = 8, 8/2 = 9, 8/5 = 3.6, 8/.8,. max units SUPL-4 25 mm : 25X5=25, 25X2= 5, 25, 25/2= 25, 25/= 25, 25/2 = 2.5, 25/5 = 5, 25/ = 2.5, max units 25 - mm marks Independent variable is plotted on X-axis Prepare BEER'S LAW PLOT BY HAND! Use rules in SUPL-4 7. Determine SLOPE of best straight line passing through the origin vs Concentration Red Dye #2 at 47 nm Concentration (mg/l) 8. Finally, using Beer s Law, and the slope, determine concentration of an unknown solution of the same dye whose spectrum you have studied. c = abs / slope 27 ZERO ADJUSTMENT: sets % transmittance calibrates detector Needs only Periodic checking after warm-up WAVELENGTH ADJUSTMENT: sets wavelength (color) LIGHT CONTROL: sets % transmittance Needs readjustment before every measurement of a sample. (Using BLANK) 28 Some elements of common sense in using the spectrophotometer Transmittance can never be > % (Abs< ) If you get such a reading, check the amplifier adjustment. % transmittance setting may have drifted. Sometimes air bubbles form in BLANK (or in the dye solution) Cuvettes should always be aligned the same way in the sample compartment Look for a mark on the cuvette. The mark should face front of the spectrophotometer 29 DILUTION CALCULATIONS Need to prepare accurate dilutions of stock solutions Concentration = Amount / Volume (e.g.,mg/l) Amount (mg) = Concentration (mg / L) X Volume ( L) Start: Volume = V at Conc Add some solvent End: Volume = V 2 at Conc 2 What is Conc 2? Have added no additional reagent, so AMT 2 = AMT Conc 2 x V 2 Conc x V = V Conc 2 = Conc x V 2 3 5

6 9//22 E.g., 25 ml of a solution with a concentration of 3 mg/l of Red 3 is diluted to 75 ml (by adding 5 ml of distilled water). What is the final concentration of Red 3? NEXT LECTURE PAPER CHROMATOGRAPHY OF FOOD DYES Do Pre-Lab for SUSB 9 (Hand in at beginning of lab period) V Conc 2 = Conc x ml Final Conc = 3 mg/l x = mg/l 75 ml V