This document describes chromatographic separation methods for analyzing curcumin from turmeric and dyes from grape soda. It outlines a procedure using thin-layer chromatography to isolate curcumin from a turmeric extract using dichloromethane as the solvent. It also details a process employing reverse-phase chromatography on a C-18 cartridge to separate the red and blue dyes from grape soda using isopropyl alcohol solutions of varying concentrations as eluents. Optical densities are measured to determine the amounts of each dye in a can of grape soda.

1. CHROMATOGRAPHIC SEPARATION METHODS
SEPARATION BY THIN-LAYER CHROMATOGRAPHY (TLC)
&
REVERSE-PHASE

2. CURCUMIN, ANYONE ?
1,529,560 New Cancer Cases in 2010
569,490 Cancer Deaths in 2010
Curcumin Prevents Cancer, Heart Disease,
Alzheimer’s Disease and Obesity.
Cancer Cell

3. PART 1: Thin Layer Chromatography Analysis of CURCUMIN in Turmeric
Purpose:
ISOLATION OF THE YELLOW PIGMENT
CURCUMIN FOUND IN THE SPICE TUMERIC
BY THIN-LAYER CHROMATOGRAPHY
(TLC) METHOD & USING A POLAR
ORGANIC SOLVENT. Curcumin
Turmeric Root
TUMERIC COMES FROM THE CURCUMA LONGA
PLANT (GINGER-LIKE) FOUND IN ASIA.
Cell Menbrane: Phospholipid Bi­layer

4. PART 1: Thin Layer Chromatography Analysis of CURCUMIN in Turmeric
Step #1: Heating Reflux
60 G OF STORE-BOUGHT
TUMERIC SPICE MIXED WITH
150 ML DICHLOROMETHANE IN Water Out
A FLASK
Condenser
REFLUX APPARATUS Water In
Flask
CONNECTION TO COLD WATER Sink Outlet
Hot Water
HEATED FOR 3 HOURS IN HOT Hot Plate
WATER BATH (45°C)

5. PART 1: Thin Layer Chromatography Analysis of CURCUMIN in Turmeric
Step #2: Suction Filtration
Funnel
VACUUM PUMP IS Rubber Stopper
CONNECTED TO A FLASK
Plastic Tubing
WITH THE RIGHT SIZE
TUBING
PAPER FILTER IS PLACED
INSIDE THE FUNNEL Flask
Vacuum Pump
FUNNEL IS SECURED INTO
THE FLASK BY A RUBBER
STOPPER

6. PART 1: Thin Layer Chromatography Analysis of CURCUMIN in Turmeric
Step #3: TLC Analysis
DROPPED 3 ML OF SOLVENT (5% METHANOL IN
DICHLOROMETHANE) INTO BEAKER #1
SPOTTED A TLC PLATE WITH TURMERIC EXTRACT
TLC PLATE IN BEAKER COVERED WITH WATCH GLASS Beaker #1
PREPARED 2ND BEAKER WITH SAME SOLVENT AS
ABOVE BUT ADDED 3 DROPS OF METHANOL (INCREASE
POLARITY)
PREPARED PLATE #2 & REPEATED
THE PLATES ARE TAKEN OUT AS SOON AS THE
SOLVENT IS 1CM BELOW THE TOP OF THE PLATE
Beaker #2

7. PART 1: Thin Layer Chromatography Analysis of CURCUMIN in Turmeric
Step #3: Results of TLC Analysis
CURCUMIN TRAVELED ON PLATE #2, SOLVENT
FROM THE BOTTOM UP TRAVELED 8.10 CM
ON PLATE #1, SOLVENT THE RF FOR 1ST SPOT
TRAVELED 8.05 CM FROM BOTTOM UP IS 0.48
THE RF FOR 1ST SPOT THE RF FOR 2ND SPOT
FROM BOTTOM UP IS 0.30 FROM BOTTOM UP IS 0.67
THE RF FOR 2ND SPOT THE RF FOR 3RD SPOT
FROM BOTTOM UP IS 0.56 FROM BOTTOM UP IS 0.86
THE DARKEST SPOT THE DARKEST SPOT
(2ND SPOT) IS CURCUMIN. (3RD SPOT) IS CURCUMIN.
Rf= distance traveled by spot
distance traveled by solvent
TLC Plate #1 TLC Plate #2

8. EVER WONDERED WHAT IS IN YOUR GRAPE SODA?
2 SYNTHETIC DYES:
Allura Red AC
Brilliant Blue FCF
Allura Red AC can be found in
tattoo inks, cosmetics, children's medications, cotton candy.
On June 30, 2010, the Center for Science in the Public Interest (CSPI)
called for the FDA to ban Red 40.
Brilliant Blue FCF is a synthetic dye made with aromatic
hydrocarbons from petroleum.
Can be found in
ice cream, canned processed peas, dairy products,
sweets, soaps, shampoos & mouthwash.
It is poorly absorbed from the gastro‐intestinal tract and
95% of the ingested dye can be found in the feces.

9. PART 2: Separation and Analysis of Dyes in Grape Soda
Purpose:
ISOLATION OF THE TWO DYES FOUND IN GRAPE SODA
USING A REVERSE-PHASE SEP-PAK C-18
CARTRIDGE.
CH
3
Cell Menbrane
CH
3
CH H C
3 3
Red Dye (FD&C #40), Allura Red Blue Dye (FD&C #1), Brilliant Blue

10. PART 2: Separation and Analysis of Dyes in Grape Soda
Step #1: Separation of the Red Dye (FD&C #40)
PRE-TREAT THE SEP-PAK CARTRIDGE WITH
10.0 ML OF 70% ISOPROPYL ALCOHOL
SOLUTION
10.0 ML OF WATER.
FORCE 5.0 ML OF GRAPE SODA THE SEP-PAK
USING A SYRINGE.
COLLECT THE 1ST FRACTION (ELUENT).

11. PART 3:Separation and Analysis of Dyes in Grape Soda
FORCE 5.0 ML 5% ISOPROPYL
ALCOHOL SOLUTION THROUGH
THE SEP-PAK.
COLLECT THE 2ND FRACTION
REPEAT 3 X. THIS YIELDS
FRACTIONS 3 THROUGH 5.

12. PART 3: Separation and Analysis of Dyes in Grape Soda
FORCE 2.5 ML 5% ISOPROPYL
ALCOHOL SOLUTION THROUGH
THE SEP-PAK.
COLLECT THE 6TH FRACTION
REPEAT 4 X. THIS YIELDS
FRACTIONS 7 THROUGH 10.

13. PART 3: Separation and Analysis of Dyes in Grape Soda
Step #2: Separation of the Blue Dye (FD&C #1)
FORCE 5.0 ML 20% ISOPROPYL
ALCOHOL SOLUTION THROUGH
THE SEP-PAK.
COLLECT THE 11TH FRACTION
REPEAT 3 X. THIS YIELDS
FRACTIONS 12 THROUGH 14.

14. PART 3: Separation and Analysis of Dyes in Grape Soda
SAMPLES’ OPTICAL
DENSITIES ARE READ WITH
A SPECTROPHOTOMETER.
AMOUNT OF DYE IN ONE CAN OF GRAPE SODA:
• 2.59 MG OF RED DYE
• 0.36 MG OF BLUE DYE

15. SOURCES
_____________________________________
http://en.wikipedia.org/wiki/Stearic_Acid
http://qwickstep.com/search/sodium-chloride-in-water.html
http://www.3dchem.com/molecules.asp?ID=176
http://www.btinternet.com/~martin.chaplin/molecule.html
http://www.worldofmolecules.com/3D/turmeric_3d.htm
http://upload.wikimedia.org/wikipedia/commons/6/67/Turmericroot.jpg
http://en.wikipedia.org/wiki/Allura_Red_AC
http://en.wikipedia.org/wiki/Brilliant_Blue_FCF
http://en.wikipedia.org/wiki/Allura_Red_AC
http://en.wikipedia.org/wiki/Brilliant_Blue_FCF
http://www.northallegheny.org
http://nih.gov/
http://drvikram.com/herbal‐products.htm
http://www.madhealth.net/curcumin‐prevents‐cancer‐heart‐disease‐
alzheimer%E2%80%99s‐disease‐and‐obesity/
http://www.themoneytimes.com/files/cancer%20cells.jpg

16. THANK YOU TO PROFESSOR
KIMBERLY STIEGLITZ
_______________________
PRESENTED BY
HELENE GAUDETTE
FOR
SCI 124 PRINCIPLES OF CHEMISTRY II