Color. Spectrophotometry. of F o o d D y e s. & Beer’s Law. A = ε b c. Concentration. Questions. OBJECTIVES. What is the quantitative basis for the color of substances?. How is the absorption/transmission of light measured?.
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OBJECTIVES
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?
Purpose
Purpose
For a simple, transparent, COLORED material, e.g.
ROYGBV
ROYGBV
Incident white light
Transmitted light
It(λ)
Ii(λ)
VIOLET CLEAR SOLUTION
Can plot a graph showing the percent of light of each discrete COLOR (wavelength, λ) transmitted by the solution
It(λ)
Percent
Transmittance
100 X
%T(λ) =
Ii(λ)
Or, instead, we can plot the percent of light absorbed
% Transmitted
Wavelength (nm)
← UV
IR
Violet
Blue
Green
Yellow
Red
% Absorbed = 100%  % Transmitted
% Absorbed
Violet
Blue
Yellow
Red
Green
VISIBLE AND ULTRAVIOLET:
Electronic structure of molecules
λ= 100 nm – 800 nm
INFRARED REGION:
Vibrations of molecules
800 nm – 100 m
nm = nano meter = 109 m
What determines INTENSITY of absorption?
EFFICIENCY of INTERACTION between Molecules & Electromagnetic Radiation
ASPECTROPHOTOMETER measures the RELATIVEabsorption of light of DIFFERENT WAVELENGTHS by a substance
SOURCE of light,
MONOCHROMATOR– an element to select particular wavelengths
SAMPLE & CONTAINER
DETECTOR to measure intensity of light transmitted through sample, and
ELECTRONICS to translate detector
output into numbers
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 )
[Water]
[None]
e.g., a solvent, a container, another colored substance etc.

=
E.g., the rods and cones of the human eye
% Absorbance
% Absorbance
% Absorbance
BLANK + SAMPLE
BLANK
SAMPLE
This insures that measured Absorbance 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?
BEERLAMBERT LAW (1760  1852)
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 bc
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=CONCENTRATIONof the absorbing substance
−−−−
It
Definition of Absorbance
Smaller It means larger A
A
=
log
It
−−−−
I0
How is Absorbance related to %Transmittance?
I0
It
%T
=
100
100
A = log −−−−
%T
= log 100  log %T
A = 2.000…  log %T
Beer’s Law states
ABSORBANCE
is proportional to concentration
B= False
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.
What do we need to know to relate
Absorbance to concentration?
Need to know:
ABSORPTIVITY, ε , AND
THICKNESS OF THE CUVETTE,b
OR
at least,
their product, εb
The name given to the sample cells that we will use
How do we proceed experimentally?
1.) Determine the substance’s SPECTRUM in the desired wavelength range – i.e. absorbance as a function of wavelength
2.) Find WAVELENGTH at which the substance has the LARGEST ABSORBANCE
(lowest%Transmittance)
The Absorbance will be most sensitive
to concentration at this wavelength
This wavelength is called the
ANALYTICAL WAVELENGTH
2.) Then we prepareseries of solutions of
KNOWNCONCENTRATIONSof the substance
c/8 c/4 c/2 c
3.)MeasureABSORBANCEof each solution at itsANALYTICAL WAVELENGTH
SLOPE =
/
Abs = log (I0/I)
= 0.0263 L/mg
25.0
25.0
0.658
0.658
Note : Absorbance is dimensionless.
BLANK
20
Slope of Beer’sLaw plot is ε X b, where
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 = 0.0263 X C (where C is in mg/L)
Unitsof the slope of the Beer’s Law plotare (always) reciprocal concentration(e.g., L / mg)
A Beer’s 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...
Abs = 0.0263 X c (c in mg/L)
Abs = 0.263
c = 0.263 / 0.0263 = 10.0 mg / L
C 10.0 mg / L
In this exercise, we examine spectrophotometric properties of
7 FOODDYES
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
Yellow 6
WORK IN PAIRS
2. Determine SPECTRUM
(abs vs) of diluted stock
solution between
350 nm and 750 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, 1/2, 1/4, 1/8, and 1/16
5. Determine ABSORBANCEof stock solution & dilutions at analytical wavelength
Why can’t you make Beer’s Law absorbance measurements before determining spectrum?
2. All pairs begin by preparing dilutionsfor Beer’s Law
While doing that:
TAs send one pair at the time to the recording spectrophotometerto obtain spectrum of the dye.
2 PAIRSwillSHAREONE SPECTRONIC 20
In Part 2 (Beer’s Law)
This process avoids resetting spectrophotometer after each measurement
Do NOT try to interleave two groups’ measurements
Purpose
dependent
landscape
portrait
independent
SUPL004
1 Box (mm) must represent a multiple or submultiple of 1, 2 or 5 units (not 3 or 4)
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
180  1 mm marks
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
Independent variable is plotted on
Xaxis
250  1 mm marks
BEER'S LAW PLOT
BY HAND!
Use rules in SUPL004
7. Determine SLOPE of best straight line passing through the origin.
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
sets wavelength (color)
ZERO ADJUSTMENT:
sets 0% transmittance – calibrates detector
Needs only Periodic checking
after warmup
LIGHT CONTROL:
sets 100 % transmittance
Needs readjustment before every measurement of a sample. (Using BLANK)
may have drifted.
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 = V1 at Conc1
Add some solvent
End: Volume = V2 at Conc2
What is Conc2?
Have added no additional reagent, so
AMT2 = AMT1

Conc2 x V2

Conc1 x V1
=
V2
C2
V1
Conc2 = Conc1 x V2
V1
C1
25 mL of a solution with a concentration of
30 mg/L of Red 3 is diluted to 75 mL
(by adding 50 mL ofdistilled water).
What is the final concentration of Red 3?
V1
Conc2 = Conc1 x  V2
25 mL
Final Conc=30 mg/Lx= 10 mg/L
75 mL
What is the concentration of a solution made by diluting 15.0 mL of a 2.50 M solution to 150. mL?
15.0 mL
Conc2=2.50Mx=0.250 M
150 mL
A0.250 M
PAPER CHROMATOGRAPHY OF FOOD DYES
Read SUSB – 009
Do PreLab for SUSB – 009
(Hand in at beginning of lab period)
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