Separation and Identification of Food Dyes by

2. Separation and Identification of Food Dyes by Chromatography (A TEST EXERCISE - 105 points)

3. OBJECTIVES Learning: What properties can be the basis for separation of mixtures ? What is the quantitative basis for chromatographicseparation What experimental conditions optimize chromatographic separation? 105 pts Performance/Assessment: With what accuracy can you identify the food dyes in an unknown mixture by paper chromatography?

4. Concepts: • capillary action chromatography • components interactions • mobile phase migration rate • mixture origin • rates resolution • retention factor solvent front • solvents spot • stationary phase

5. Techniques: • preparing chromatogram • optimizing resolution by solvent choice • measuring spots • Apparatus: • solvent chamber (beaker/watch glass) • ruler • Chromatogram • toothpick

6. Background How are substances separated from one another? Separation Techniques Decantation Crystallization Distillation Evaporation Flotation Extraction Magnetic Separation Chromatography Precipitation Sieving Sublimation Zone Refining Sedimentation Drying Centrifugation Electrophoresis ……..

7. Background Background 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: Column, gas phase, gel, paper, etc. In this exercise: STATIONARY PHASE = Chromatography paper MOBILE PHASES = Solvents (H2O, etc.) The mobile phases pass through the stationary phase by CAPILLARY ACTION

8. Capillary Action CAPILLARY ACTION: Spontaneous rising or lowering of a liquid level in a narrow tube Caused by a BALANCE BETWEEN COHESIVE FORCES ADHESIVE FORCES and Those that keep a liquid together and try to minimize its surface area Forces of attraction (or repulsion) between a liquid and its container

9. Water & Hg Consider Behavior of Water and Mercury with respect to Glass Glass Tube H2O Hg 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

10. How does chromatographic separation work? QUALITATIVELY: Dye Different solute molecules interact differently with the stationary phase , and Paper have varying solubility in mobile phase Solvent The interactions can be based on: Polarity Bonding Molecular size Electrical Charge etc. Therefore, DIFFERENT SUBSTANCESgenerally haveDIFFERENT INTERACTIONS

11. 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 Three water-like hydroxyl units can HYDROGENBOND to water or to IONIC dye molecules

12. The Dyes YELLOW 6 452 RED 40 496 BLUE 1 793 GREEN 3 809

13. BLUE 2466 YELLOW 5 534 Na+ RED 3 880 Na+ 3’, 6’ -dihydroxy 2’, 4’, 5’, 7’ – tetra iodo spiro [isobenzofuran-1(3H), 9’ – [9H] xanthen] -3-one disodium salt

14. The Experimental Arrangement Solvent Chamber Paper Chromatograph Solvent rises up paper through capillary interaction Solvent Initial Dye Spot Origin Line

15. Solvent Front 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

16. Migration Rates - Quantitative Solvent Front For a given: dsolvent • component, • stationary phase, and dspot • mobile phase, we can • define the: All distances are measured from the ORIGIN Origin Solvent Level • Rf (Retention Factor) Distance moved by COMPONENT Rf = −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Distance SOLVENT FRONT has moved TheRELATIVE MIGRATION RATE and therefore, Rf for a given substance with a given stationary phase and given solvent is REPRODUCIBLE

17. The Rf of the spot on the chromatogram shown to the left is: Solvent Front • 1.32 • 0.756 • 0.756 cm 9.72 cm 7.35 cm Origin

18. Solvent Front The Rf of the spot on the chromatogram shown to the left is: > 1 A 1.32 B 0.756 C. 0.756 cm X 9.72 cm X 7.35 cm Origin

19. Solvent Front The Rf of the spot on the chromatogram shown to the left is: 9.72 cm 7.35 Rf= ———— = 0.756 9.72 7.35 cm Origin

20. The mobile phase also affects migration rates (i.e, Rf values) • In this exercise, we will work with: • a FIXED stationary phase (Chromatography Paper) • 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 Ideally, we seek a SINGLE solvent that clearly disperses ( RESOLVES ) all seven dyes most effectively. (Pre-lab Exercise #3) * A polarsolvent is one which tends to dissolve ionic substances better than molecular substances

21. Procedure 1. Working in groups of three You will be given authentic samples of the 7 FDA dyes and three solvents PART 1 ONLY! Salt Water Isopropyl Alcohol Water 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

22. Smith, J H2O/NaCl The Chromatogram for Part 1 X1 = R40 + G3 X3 = R3 + B2 Show Solvent YOUR CHOICE X2 = B1 + Y5 What about the 4 remaining locations?

23. Chromatogram Layout Dimensions We want to be able to track 7 + 4 = 11 spots X cm X 12 cm …. 1 cm

24. 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 TOOTHPICKS

26. Construction of the Paper Cylinder Chromatogram is rolled and stapled into a cylinder. Stapled edges must not overlap or be touching.

27. Place cylindrical chromatogram in solvent chamber. Chromatogram should not touch walls of chamber. Replace cover on solvent chamber as quickly as possible. Solvent will rise through the chromatogram. Observe the SOLVENT FRONT carefully.

28. If the cylinder edges overlap Instead of You will get

29. 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 beforesolvent front becomes • invisible, trace its location with pencil. Measure Rf’sof 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.

30. Chromatogram should be removed from solvent chamber • When solvent front reaches top of paper • When solvent front is 1½ cm from origin • When solvent front is 1½ cm from top • When first spot is 1½ cm from top

31. Chromatogram should be removed from solvent chamber C= When solvent front is 1½ cm from top

32. WORKINGALONE FROM THIS POINT FORWARD! WORKING ALONE FROM THIS POINT FORWARD! PART 2 - UNKNOWN You 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 increasingconcentration. 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 e.g., 2 toothpicks-ful on spot X1, 3 toothpicks-ful on spot X2, 4 toothpicks-ful on spot X3 yellow AND

33. 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

34. The Chromatogram for Parts 2a & 2b Unknown conc1 Unknown conc3 Show Solvent & Unk # Unknown conc2 Food Sample

35. 137 Place unknown sticker here B2, R3, Y6 Be sure to list dyes you have found in space provided

36. Data Sheet for Unknown

37. The data sheet provides for reporting 4 unknowns because each groupmust report 4 dyesin their unknown. • True • False

38. The data sheet provides for reporting 4 unknowns because each groupmust report 4 dyesin their unknown. Each student will be given an unknown. It may contain 1 or 2 or 3 or 4 of the food dyes B= False

39. 60 X 50 M&Mixels M & M’s as an art medium http://www.research.ibm.com/ed/ed_siggraph01.htm

40. Note the curved solvent front A Typical Chromatogram

41. 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.

42. ITEMS NEEDING PARTICULAR CARE • Preparation of solvent containers • Do this FIRST to permit solvent vapors to fill container and equilibrate • Prevents solvent evaporation during development of chromatogram • Preparation of chromatography paper • Handle carefully with clean hands • Oils interfere with capillary paths • Draw origin lines and marks ( in PENCIL ) • As instructed

43. 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

44. 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 inserted • To maintain solvent vapor equilibrium

45. 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 Rfdetermination. 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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47. Lab Expectations Lab Expectations • 1.Read Exercise and other assigned readings • 2.AttemptPre-lab exercise • Check Web for helpful supplements • 3.Attend pre-lab lecture • Re-attempt Pre-lab exercise • Seek help in help room if necessary • 4.FinishPre-lab exercise • 5.Attend Laboratory • Turn in Pre-lab exercise • Perform Exercise recording activities in lab notebook • Transcribe final data onto data sheets • Turn in data sheet with execution plan

48. FOR NEXT LECTURE READ & DO PRE LAB - SUSB-030 PREPARATION OF ALUM

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