1 Spectrophotometry & Beer’s Law A = ε b c of Food Dyes 2.

Spectrophotometry & Beers Law A = b c of Food Dyes 2

How is the absorption/transmission of light measured? How is the intensity of color related to the concentration of colored solutions? What is the quantitative basis for the color of substances? OBJECTIVES What considerations go into graphing experimental data? Questions 3

Concepts, Techniques Concepts: AbsorbanceAbsorption AbsorptivityAnalytical Wavelength Beers LawBlank ConcentrationElectromagnetic Spectrum IncidentIntensity SpectroscopySpectrum Transmittance Wavelength Purpose 4

Concepts, Techniques Techniques: Visible Spectroscopy Quantitative dilution Graphing Plotting /Curve fitting Purpose Apparatus: Recording Spectrophotometer Spectronic 20 Pipet/SyringeCuvette 5

For a simple, transparent, COLORED material, e.g. Can plot a graph showing the percent of light of each discrete COLOR (wavelength, ) transmitted by the solution Transmitted light VIOLET VIOLET CLEAR SOLUTION Incident white light It()It() Ii()Ii() It()It() 100 X Ii()Ii() %T( ) = ROYGBVROYGBV ROYGBV Percent Transmittance 6 BACKGROUND

Violet Red Or, instead, we can plot the percent of light absorbed GreenYellowBlue % Transmitted UV IR Wavelength (nm) 7 % Transmittance of White Light by a Violet Solution

VioletRedBlue Green % Absorbed Yellow % Absorbed = 100% - % Transmitted 8 % Absorbed of White Light by a Violet Solution

VISIBLE AND ULTRAVIOLET : Electronic structure of molecules INFRARED REGION: Vibrations of molecules What determines INTENSITY of absorption? EFFICIENCY of INTERACTION between Molecules & Electromagnetic Radiation = 100 nm 800 nm 800 nm 100 m nm = nano meter = 10 -9 m 9 What determines the WAVELENGTHS of light that substances absorb?

A SPECTROPHOTOMETER measures the RELATIVE absorption of light of DIFFERENT WAVELENGTHS by a substance DETECTOR to measure intensity of light transmitted through sample, and ELECTRONICS to translate detector output into numbers SOURCE of light, MONOCHROMATOR an element to select particular wavelengths SAMPLE & CONTAINER 10 Which DEVICE measures this phenomenon?

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 This insures that measured Absorbance of light is due only to the substance of interest BLANKBLANK + SAMPLESAMPLE - = BLANK ( a BLANK ) Sample container Solvent Other reagents, etc. [ Water ] [None] % Absorbance e.g., a solvent, a container, another colored substance etc. 11 E.g., the rods and cones of the human eye

How is the amount of COLOR absorbed by a chemical species related to the BEER-LAMBERT LAW (1760 - 1852) constant absorbance = constant x path length x concentration Amount of light absorbed by a transparent solution is proportional to the thickness of the solution & concentration of the absorbing species A = b c wavelength A = ABSORBANCE at a specified wavelength constant wavelength = 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 CONCENTRATION of that species? 12

A %T= log I 0 I t I 0 100 A = log %T = log 100 - log %T = How is Absorbance related to %Transmittance? A = 2.000 - log %T Definition of Absorbance Smaller I t means larger A I0I0 ItIt 13

Beers Law states that % transmittance is proportional to path length & concentration 14 A.True B.False

Beers Law statesABSORBANCE is proportional to concentration A = b c B = False 15 1. Memory 2. Common Sense: A solution with larger concentration must be darker &, therefore, absorb more and transmit less light! I.e., have a smaller %T.

A = b c Need to know: OR at least, their product, b What do we need to know to relate Absorbance to concentration? The name given to the sample cells that we will use ABSORPTIVITY, , AND THICKNESS OF THE CUVETTE, b 16

2.) Find WAVELENGTH at which the substance has the LARGEST ABSORBANCE (lowest %Transmittance) How do we proceed experimentally? The Absorbance will be most sensitive to concentration at this wavelength This wavelength is called the ANALYTICAL WAVELENGTH 1.) Determine the substances SPECTRUM in the desired wavelength range i.e. absorbance as a function of wavelength 17

2.) Then we prepare series of solutions of KNOWN KNOWN CONCENTRATIONS of the substance 3.) Measure ABSORBANCE of each solution at its ANALYTICAL WAVELENGTH c/8 c/4c/2 c 18

Conc (mg/L)Abs 0.00.000 9.70.29 21.00.52 29.60.75 40.01.05 4.) Plot ABSORBANCE vs CONCENTRATION Tabulate the data and BLANK 19

20 BLANK SLOPE = 25.0 0.658 / = 0.0263 L/mg SUPL-004 25.0 0.658 20 Abs = log (I 0 /I) Note : Absorbance is dimensionless.

Beers Law expression for Red #2 at its analytical wavelength in this cell becomes: A = 0.0263 X C (where C is in mg/L) Slope of Beers Law plot is X b, where = 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 & b separately. Units of the slope of the Beers Law plot are (always) reciprocal concentration (e.g., L / mg) 21

A Beers Law plot has a slope of 0.0263 L / mg. The absorbance of a solution is measured to be 0.263. What is the concentration of the solution? Please make your selection... 22 A. 0.100 mg / L B. 1.00 mg / L C. 10.0 mg / L D.100. mg / L E.It depends on the blank

Abs = 0.0263 X c (c in mg/L) c = 0.263 / 0.0263 = 10.0 mg / L Slope = 0.0263 L / mg C10.0 mg / L Abs = 0.263 23

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 1, Blue 2, Green 3, Yellow 5, Yellow 6, Red 3, and Red 40 We will further examine dyes and their chemical properties next week 24

Blue 2 Yellow 6 25

1.Pair obtains 50 mL of ONE assigned assigned dye solution PROCEDURE WORK IN PAIRS Using recording spectrophotometer 2. Determine SPECTRUM (abs vs ) of diluted stock solution between 350 nm and 750 nm 26

4. Prepare dilutions of stock solution of precisely known concentration 3/4, 1/2, 1/4, 1/8, and 1/16 5. Determine ABSORBANCE of stock solution & dilutions at analytical wavelength most dilute most dilute solution (1/16) first, 3. Determine ANALYTICAL WAVELENGTH, i.e., wavelength of maximum absorbance. next most dilute next most dilute solution (1/8), etc. 27

preparing dilutions 2. All pairs begin by preparing dilutions for Beers Law Procedure Schedule 28 ONE dye 1. You will be assigned a partner and ONE dye While doing that: one pair at the time TAs send one pair at the time to the recording spectrophotometer to obtain spectrum of the dye. Why cant you make Beers Law absorbance measurements before determining spectrum?

In part 2 (Beers Law - Groups of two should use spectrophotometers in series i.e., group 1 does a complete Beers Law data acquisition sets wavelength check blank measure dye1 / conc 1 measure dye1 / conc 2, etc. When group 1 finishes, group 2 resets Spectronic 20 and does the same for its dye In Part 2 (Beers Law) 2 PAIRS will SHARE ONE SPECTRONIC 20 This process avoids resetting spectrophotometer after each measurement 29 Do NOT try to interleave two groups measurements

6. Prepare BEER'S LAW PLOT BY HAND! Use rules in SUPL-004 30

Concepts, Techniques Concepts - Graphing: AbscissaOrdinate ScaleSlope InterceptIndependent Variable Dependent VariableGrid PortraitLandscape OrientationRange GraphGrid Lines Purpose 31

SUPL-004 Summary 32 The principal rules for drawing graphs described in SUSB-004 are: The abscissa (x-axis) shall generally represent the independent variable. The dependent variable is therefore represented by the ordinate (y-axis) The scales shall use 1, 2, or 5 units or decimal submultiples or multiples thereof per mm. I.e., 1 mm = 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, units The orientation (portrait or landscape) shall be chosen to maximize the plot area independent dependent portrait landscape

33 250 mm : 250X5=1250, 250X2= 500, 250, 250/2= 125, 250/10= 25, 250/20 = 12.5, 250/50 = 5, 250/100 = 2.5, max units 180 mm: 180, 180/2 = 90, 180/5 = 36, 180/10 = 18, 180/20 = 9, 180/50 = 3.6, 180/100 =1.8, . max units 250 - 1 mm marks 180 - 1 mm marks Independent variable is plotted on X-axis Portrait or Landscape? 1 Box (mm) must represent a multiple or submultiple of 1, 2 or 5 units (not 3 or 4) SUPL-004

slope 8. Finally, using Beers Law, and the slope, determine concentration of an unknown solution of the same dye whose spectrum you have studied. 6. Prepare BEER'S LAW PLOT 7. Determine SLOPE of best straight line passing through the origin. BY HAND! c = abs / slope Use rules in SUPL-004 34

THE SPECTROPHOTOMETER 35

WAVELENGTH ADJUSTMENT: sets wavelength (color) ZERO ADJUSTMENT: sets 0% transmittance calibrates detector Needs only Periodic checking after warm-up LIGHT CONTROL: sets 100 % transmittance Needs readjustment before every measurement of a sample. (Using BLANK) 36

Some Spectrophotometers have an analog (meter) readout 38

Some elements of common sense in using the spectrophotometer Transmittance can never be > 100% (Abs< 0) If you get such a reading, check the amplifier adjustment. 0% 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 39

Have added no additional reagent, so AMT 2 = AMT 1 DILUTION CALCULATIONS Need to prepare accurate dilutions of stock solutions Concentration = Amount / Volume (e.g.,mg/L) Start: Volume = V 1 at Conc 1 End: Volume = V 2 at Conc 2 What is Conc 2 ? V 1 Conc 2 = Conc 1 x ------ V 2 || Conc 2 x V 2 || Conc 1 x V 1 = Add some solvent Amount (mg) = Concentration (mg / L) X Volume ( L) V1C1V1C1 V2C2V2C2 40

E.g., 25 mL of a solution with a concentration of 30 mg/L of Red 3 is diluted to 75 mL (by adding 50 mL of distilled water). What is the final concentration of Red 3? 25 mL Final Conc = 30 mg/L x -------- = 10 mg/L 75 mL V 1 Conc 2 = Conc 1 x --- V 2 41

What is the concentration of a solution made by diluting 15.0 mL of a 2.50 M solution to 150. mL? 42 A.0.250 M B.1.50 M C.25.0 M

15.0 mL Conc 2 = 2.50M x ---------- = 0.250 M 150 mL A0.250 M 43 What is the concentration of a solution made by diluting 15.0 mL of a 2.50 M solution to 150. mL?

PAPER CHROMATOGRAPHY OF FOOD DYES Read SUSB 009 Do Pre-Lab for SUSB 009 (Hand in at beginning of lab period) 44 NEXT EXERCISE

ANY ? QUESTIONS 45

NO ADMITTANCE TO THE LAB WITHOUT SAFETY GOGGLES Gorilla 5 point penalty 47

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1 Spectrophotometry & Beer’s Law A = ε b c of Food Dyes 2.

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Transcript of 1 Spectrophotometry & Beer’s Law A = ε b c of Food Dyes 2.