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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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spectrophotometry

Color

Spectrophotometry

of FoodDyes

&

Beer’s Law

A= ε bc

Concentration

questions
Questions

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?

concepts techniques
Concepts, Techniques
  • Concepts:
    • Absorbance Absorption
    • Absorptivity Analytical Wavelength
    • Beer’s Law Blank
    • Concentration Electromagnetic Spectrum
    • Incident Intensity
    • Spectroscopy Spectrum
    • Transmittance Wavelength

Purpose

concepts techniques1
Concepts, Techniques
  • Techniques:
    • Visible Spectroscopy Quantitative dilution
    • Graphing Plotting /Curve fitting
  • Apparatus:
    • Recording Spectrophotometer
    • Spectronic 20
    • Pipet/Syringe Cuvette

Purpose

background
BACKGROUND

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)

transmittance of white light by a violet solution
% Transmittance of White Light by a Violet Solution

Or, instead, we can plot the percent of light absorbed

% Transmitted

Wavelength (nm)

← UV

IR 

Violet

Blue

Green

Yellow

Red

absorbed of white light by a violet solution
% Absorbed of White Light by a VioletSolution

% Absorbed = 100% - % Transmitted

% Absorbed

Violet

Blue

Yellow

Red

Green

what determines the wavelengths of light that substances absorb
What determines the WAVELENGTHS of light that substances absorb?

VISIBLE AND ULTRAVIOLET:

Electronic structure of molecules

λ= 100 nm – 800 nm

INFRARED REGION:

Vibrations of molecules

800 nm – 100 m

nm = nano meter = 10-9 m

What determines INTENSITY of absorption?

EFFICIENCY of INTERACTION between Molecules & Electromagnetic Radiation

which device measures this phenomenon
Which DEVICE measures this phenomenon?

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

slide11

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.

[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

slide12

How is the amount of COLOR absorbed by a chemical species related to the

CONCENTRATION of that species?

BEER-LAMBERT 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

slide13

I0

−−−−

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

slide15

A= ε bc

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.

slide16

A= ε bc

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

slide17

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

slide18

2.) Then we prepareseries of solutions of

KNOWNCONCENTRATIONSof the substance

c/8 c/4 c/2 c

3.)MeasureABSORBANCEof each solution at itsANALYTICAL WAVELENGTH

slide19

Tabulate the data

BLANK

and

4.) Plot ABSORBANCEvsCONCENTRATION

slide20

SUPL-004

SLOPE =

/

Abs = log (I0/I)

= 0.0263 L/mg

25.0

25.0

0.658

0.658

Note : Absorbance is dimensionless.

BLANK

20

slide21

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)

slide22

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...

  • 0.100 mg / L
  • 1.00 mg / L
  • 10.0 mg / L
  • 100. mg / L
  • It depends on the blank
slide23

Slope = 0.0263 L / mg

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

slide24

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

slide25

Blue 2

Yellow 6

slide26

PROCEDURE

WORK IN PAIRS

  • Pair obtains 50 mL of ONE
  • assigned dye solution

2. Determine SPECTRUM

(abs vs) of diluted stock

solution between

350 nm and 750 nm

Using recording spectrophotometer

slide27

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

  • most dilute solution (1/16) first,
  • next most dilute solution (1/8), etc.
slide28

Procedure Schedule

Why can’t you make Beer’s Law absorbance measurements before determining spectrum?

  • You will be assigned a partner and ONE dye

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.

slide29

2 PAIRSwillSHAREONE SPECTRONIC 20

In Part 2 (Beer’s Law)

  • In part 2 (Beer’s Law - Groups of two should use spectrophotometers in series
  • i.e., group 1 does a completeBeer’s Law data acquisition
    • sets wavelength
    • check blank
    • measure dye1 / conc1
    • measure dye1 / conc2, etc.
  • When group 1finishes,
    • group 2 resets Spectronic 20 and does the same for its dye

This process avoids resetting spectrophotometer after each measurement

Do NOT try to interleave two groups’ measurements

slide30

6. Prepare

BEER'S LAW PLOT

BY HAND!

Use rules in SUPL-004

concepts techniques2
Concepts, Techniques
  • Concepts - Graphing:
    • Abscissa Ordinate
    • Scale Slope
    • Intercept Independent Variable
    • Dependent Variable Grid
    • Portrait Landscape
    • Orientation Range
    • Graph Grid Lines

Purpose

supl 004 summary
SUPL-004 Summary
  • 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

dependent

landscape

portrait

independent

slide33

Portrait or Landscape?

SUPL-004

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

X-axis

250 - 1 mm marks

slide34

6. Prepare

BEER'S LAW PLOT

BY HAND!

Use rules in SUPL-004

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

slide36

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)

some elements of common sense in using the spectrophotometer
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
slide40

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 = 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

slide41

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 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
What is the concentration of a solution made by diluting 15.0 mL of a 2.50 M solution to 150. mL?
  • 0.250 M
  • 1.50 M
  • 25.0 M
slide43

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

slide44

NEXT EXERCISE

PAPER CHROMATOGRAPHY OF FOOD DYES

Read SUSB – 009

Do Pre-Lab for SUSB – 009

(Hand in at beginning of lab period)

slide45

ANY

?

QUESTIONS

gorilla
Gorilla

5 point penalty

NO ADMITTANCE TO THE LAB WITHOUT SAFETY GOGGLES

what help is available
What Help is Available?

Lecture Notes

Flow Charts

Concept Maps

Web Supplements

Lecture Videos

Help Sessions

Faculty Office Hours