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2

Separation and Identification of Food

Dyes by

(A TEST EXERCISE - 105 points)

ELUATE

BAND 2

BAND 1

N N

NN

-O3SOH

-OOCSO3

-

3

What is the quantitative basis for chromatographic separation

What experimental conditions optimize chromatographic separation?

What properties can be the basis for separation of mixtures ?

OBJECTIVES

With what accuracy can you identify the food dyes in an unknown mixture by

paper chromatography?

Learning:

Performance/Assessment:

105 pts

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Concepts:

capillary action chromatography

components interactions

mobile phase migration rate

mixture origin

rates resolution

retention factor solvent front

solvents spot

stationary phase

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Techniques:

preparing chromatogram

optimizing resolution by solvent choice

measuring spots

Apparatus:

solvent chamber (beaker/watch glass)

ruler

Chromatogram

toothpick

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Background

DecantationCrystallizationDistillationEvaporationFlotationExtractionMagnetic SeparationChromatography

PrecipitationSievingSublimationZone RefiningSedimentationDryingCentrifugationElectrophoresis……..

Separation

How are substances separated from one another?

Techniques

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The mobile phases pass through the stationary phase by CAPILLARY ACTION

Chromatographic separation involves TWO PHASES, (i.e., states of matter).

MOBILE PHASE (gas or liquid)

STATIONARY PHASE (liquid or solid)

Technique has been developed in many different ways:

In this exercise:

MOBILE PHASES = Solvents (H2O, etc.)

STATIONARY PHASE = Chromatography paper

ELUATE

BAND 2

BAND 1

Column, gas phase, gel, paper, etc.

Background Background

CAPILLARY ACTION:Spontaneous rising or lowering of a liquid level in a narrow tube

COHESIVE FORCES

ADHESIVE FORCESand

Those that keep a liquid together and try to minimize its

surface area

Forces of attraction (or

repulsion) between a liquid and its

container

Caused by a BALANCE BETWEEN

Capillary

Action8

Consider Behavior of Water and Mercury with respect to Glass

Adhesive > Cohesive Cohesive > Adhesive

Chromatography paper can be thought of as having a large number of very small capillary channels in each of which the

liquid rises

Glass Tube

H2O Hg

Water & Hg

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How does chromatographic separation work?

QUALITATIVELY:

The interactions can be based on:

Therefore, DIFFERENT SUBSTANCES generally have DIFFERENT INTERACTIONS

DyePape

rSolvent

Different solute moleculesinteract differentlywith the stationary phase

have varying solubility in mobile phase

Molecular size Electrical Charge etc.

PolarityBonding

N N

NN

-O3SOH

-OOC

SO3-

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, and

In paper chromatography of dyes, primary basis for separation is interaction between dye molecules and paper.

Paper consists primarily of CELLULOSE, a high molecular weight polymer of the carbohydrate GLUCOSE (C6H10O5)n

O O O H O

H O C H 2

O H O

O

H O C H 2

H O O H

.....

Three water-like hydroxyl units can HYDROGEN BOND to water or to IONIC

dye molecules 11

N N

-O3S

HO

SO3-

N

-O3SCH3

OH3CN

SO3-

HO

C

N+ CH2

H5C2

SO3-

N

H5C2

SO3-

SO3-

C

N+ CH2

H5C2

SO3-

N

H5C2

SO3-

SO3-

HO

YELLOW 6 452

RED 40 496

BLUE 1 793

GREEN 3 809

The Dyes

N N

-O3S

HO

SO3-

C

N+ CH2

H5C2

SO3-

N

H5C2

SO3-

SO3-

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N

-O3SO

N

O

SO3-

H

H

N N

NN

OH

-OOC

-O3S

SO3-

O

I

O

I

C

O-

O

I

I

-O

RED 3 880

YELLOW 5 534

BLUE 2 466

3’, 6’ -dihydroxy 2’, 4’, 5’, 7’ – tetra iodo spiro

disodium salt

[isobenzofuran-1(3H), 9’ – [9H] xanthen] -3-one

Na+

Na+

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The Experimental Arrangement

Solvent Chamber

Solvent

PaperChromatograph

Initial Dye Spot

Solvent rises up paper through

capillary interaction

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Origin Line

Migration Rates - Qualitative

For a given solvent, dyes that bind to paper more tightly move

more slowly I.e., have lower

migration rates

Origin

Liquid Level

Solvent Front

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Migration Rates - Quantitative

Distance moved by COMPONENT Rf = −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

Distance SOLVENT FRONT has moved

For a given:

The RELATIVE MIGRATION RATE and therefore, Rf for a given substance with a given stationary phase and given

solvent is REPRODUCIBLE

Solvent Front

Origin

dsolvent

dspot

• component, • stationary phase, and

• mobile phase, we can define the:

• Rf (Retention Factor)Solvent Level

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All distances are measured from the

ORIGIN

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The Rf of the spot on the chromatogram shown to the

left is:

A. B. C.

0% 0%0%

Solvent Front

A. 1.32B. 0.756C. 0.756

cm

9.72 cm 7.35

cm

Origin

The Rf of the spot on the chromatogram shown to the left

is:

Solvent Front

Origin

9.72 cm

7.35 cm

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A 1.32B 0.756C. 0.756 cm

X

X

> 1

The Rf of the spot on the chromatogram shown to the left

is:

Solvent Front

Origin

9.72 cm

7.35 cm 7.35

Rf = ———— = 0.756 9.72

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In this exercise, we will work with:• a FIXED stationary phase (Chromatography Paper)

Ideally, we seek a SINGLE solvent that clearly disperses ( RESOLVES ) all seven dyes most

effectively. (Pre-lab Exercise #3)

The mobile phase also affects migration rates (i.e, Rf values)

• a RANGE of solvent mixtures of varying polarity*

Because of their differing chemical properties, the Rf’s of the seven food dyes will vary considerably from one solvent to the next

* A polar solvent is one which tends to dissolve ionic substances better than molecular substances

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Procedure

1. Working in groups of three

You will be given authentic samples of the 7 FDA dyes and three solvents

One student for each solvent !

Objective: to determine which solvent provides best separation - (i.e., RESOLUTION) of the 7 food dyes

In Part 1, Group Prepares 3 Chromatograms

PART 1 ONLY!Water Salt Water Isopropyl Alcohol

X1 = R40 + G3

X2 = B1 + Y5

X3 = R3 + B2

The Chromatogram for Part 1

ShowSolvent

Smith, J H2O/NaCl

What about the 4 remaining locations?

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YOUR CHOICE

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1 cm

X cm

X 12

cm

….

Chromatogram Layout Dimensions

We want to be able to

track 7 + 4 = 11

spots

Applying the Dye Spots on the Chromatogram

Dye spots are applied

using a toothpick

Dye spots should be small but

concentrated

Allow spots to dry before a second toothpick-ful is applied!

DON’T MIX TOOTHPICKS24

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Construction of the Paper Cylinder

Chromatogram is rolled and stapled into a cylinder.

Stapled edges must not overlap or be touching.

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Place cylindrical chromatogram in solvent chamber.

Replace cover on solvent chamber as quickly as possible.

Chromatogram should not touch walls of chamber.

Solvent will rise through the chromatogram. Observe the SOLVENT FRONT carefully.

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If the cylinder edges overlap

Instead ofYou will get

At appropriate time (when solvent front is 1½ cm from top),

• carefully remove chromatogram from solvent chamber

• watch as solvent front continues to move towards top of paper

• just before solvent front becomes invisible, trace its location with pencil.

Measure Rf’s of each of the dye spots

From three chromatograms, select solvent that gives best resolution. Be sure to give reason for your choice on data sheet.

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Chromatogram should be removed from solvent chamber

A. When solvent front reaches top of paper B. When solvent front is 1½ cm from origin C. When solvent front is 1½ cm from topD. When first spot is 1½ cm from top

A. B. C. D.

0% 0%0%0%

Chromatogram should be removed from solvent chamber

C = When solvent front is 1½ cm from top

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WORKING ALONE FROM THIS POINT FORWARD!

PART 2 - UNKNOWNYou will each be given an unknown. It may contain 2 or 3 or 4 food dyes

Prepare a chromatogram as in Part 1 with spots of each of the 7 Food Dyes.

In the three positions labeled X1, X2 and X3, you should place spots of your unknown with

This will insure that if you have light spots (e.g., the yellow dyes), you will be able to see their presence in the developed chromatogram

increasing

concentration.yellow

WORKING ALONE FROM THIS POINT FORWARD!

e.g., 2 toothpicks-ful on spot X1,

3 toothpicks-ful on spot X2,

4 toothpicks-ful on spot X3

AND

PART 3 – YOUR FOODSTUFF

In position labeled X4, place a concentrated spot of dye from your foodstuff.

If you use M&M’s or similar recommended items - simplest way to transfer dye to chromatogram is to

• wet a clean toothpick

• rub surface of M&M with wet toothpick

• transfer to chromatogram

• repeat this several times

Use solvent that gave the best resolution in Part 1

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The Chromatogram for Parts 2a & 2b

Unknownconc1

Unknownconc2

Unknownconc3

ShowSolvent& Unk #Food

Sample

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Be sure to list dyes you have found in

space provided

B2,

R3,

Y6

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Place unknown

sticker here

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Blue 1

……

Spot X1 A*Unknown @ B*2 x conc C* D*Spot X2 A*

Unknown @ B*

3 x conc C*

D*

Data Sheet for Unknown

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The data sheet provides for reporting 4 unknowns because each group must

report 4 dyes in their unknown.

A. B.

0%0%

A. TrueB. False

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The data sheet provides for reporting 4 unknowns because each group

must report 4 dyes in their unknown.

B = False

Each student will be given an unknown. It may contain 1 or 2 or 3 or 4 of the food dyes

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M & M’s as an art medium

http://www.research.ibm.com/ed/ed_siggraph01.htm

60 X 50 M&Mixels

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Note the curved solvent front

A Typical Chromatogram

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Warning!

The grading for this unknown will take into account:

• Number of dyes present and reported• Dyes present but not reported• Dyes reported but not present

The yellow dyes may be particularly

difficult to detect.

ITEMS NEEDING PARTICULAR CARE

Preparation of solvent containersDo this FIRST to permit solvent vapors to fill container and equilibrate

Prevents solvent evaporation during development of chromatogram

Preparation of chromatography paperHandle carefully with clean hands

Oils interfere with capillary pathsDraw origin lines and marks ( in PENCIL )

As instructed

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Preparation of chromatography paper

Label papers carefully at TOP Edge – small characters - in PENCIL

Pencil introduces no other dyes

Use small dye spots and make sure first spot is dry before applying second of same dye

Applying second spot while first is wet causes drop to spread

When creating cylinder, two edges of cylinder which are clipped together should not overlap

Overlap provides path other than vertical for solvent

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Preparation of chromatography paper

Liquid in container must be below origin line on paper

If not, dyes will be extracted into solvent rendering chromatogram useless

Cover container quickly after paper is insertedTo maintain solvent vapor equilibrium

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End of chromatogram development

– Remove paper when solvent front is ~1.5 cm from top edge• Solvent front continues to move after

chromatogram is removed from solvent

– When solvent front stops moving BUT BEFORE COMPLETELY DRY, mark position in pencil ( May not be a straight line)• Need solvent front for Rf determination.

Must be able to tell where it was

– Decide which part of final spots determine distance traveled by components• Spots are sometimes diffuse. Need

consistent criterion – use darkest spot

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Lab Expectations

1. Read Exercise and other assigned readings

2. Attempt Pre-lab exerciseCheck Web for helpful supplements

3. Attend pre-lab lectureRe-attempt Pre-lab exerciseSeek help in help room if necessary

4. Finish Pre-lab exercise

5. Attend Laboratory Turn in Pre-lab exercisePerform Exercise recording activities in lab notebookTranscribe final data onto data sheetsTurn in data sheet

with execution plan

Lab Expectations

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FOR NEXT LECTURE

READ & DO PRE LAB - SUSB-030

PREPARATION OF ALUM

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ANY

?QUESTIONS

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