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Slide 1
Slide 2
1
Slide 3
Spectrophotometry & Beers Law A = b c of Food Dyes 2
Slide 4
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
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
Slide 8
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
Slide 9
VioletRedBlue Green % Absorbed Yellow % Absorbed = 100% - %
Transmitted 8 % Absorbed of White Light by a Violet Solution
Slide 10
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?
Slide 11
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?
Slide 12
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
Slide 13
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
Slide 14
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
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Slide 15
Beers Law states that % transmittance is proportional to path
length & concentration 14 A.True B.False
Slide 16
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.
Slide 17
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
Slide 18
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
Slide 19
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
Slide 20
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
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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
Slide 23
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
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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
Slide 25
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
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Blue 2 Yellow 6 25
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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
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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
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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?
Slide 30
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
Slide 31
6. Prepare BEER'S LAW PLOT BY HAND! Use rules in SUPL-004
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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
Slide 34
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
Slide 35
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
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THE SPECTROPHOTOMETER 35
Slide 37
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
Slide 38
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Some Spectrophotometers have an analog (meter) readout 38
Slide 40
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
Slide 41
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
Slide 42
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
Slide 43
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
Slide 44
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?
Slide 45
PAPER CHROMATOGRAPHY OF FOOD DYES Read SUSB 009 Do Pre-Lab for
SUSB 009 (Hand in at beginning of lab period) 44 NEXT EXERCISE
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ANY ? QUESTIONS 45
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46
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NO ADMITTANCE TO THE LAB WITHOUT SAFETY GOGGLES Gorilla 5 point
penalty 47
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48 Lecture Notes Flow Charts Concept Maps Lecture Videos
Faculty Office Hours Help Sessions Web Supplements What Help is
Available?What Help is Available?