1. Observing the Effect of Polarity in the Separation of Pigments
Anish Prasanna
Jeremy Rubin
Aradhana Vyas
Block A

2. Background Used to separate mixtures Multiple types
Developed in the early 1900s

3. Problem Determine the best isopropyl alcohol to water ratio
Polarity-chromatographic separation relationship

4. Why our project is important
Chlorophyll often hides other pigments
Is a sensitive method of detection
Forensic Science

5. Paper Chromatography Introduction
Chloroplast Pigments
Chlorophyll a and b
Anthocyanin
Carotenoids
Chromatographic Separation

6. Basic Chromatography Terms
Mobile Phase
Stationary Phase
Band Broadening Theory
Solvent
Solvent Front
Solution

7. Basic Chemistry Molecular Polarity Hydrogen Bonding
Dipole-Dipole Forces
Dispersion Forces
Solubility
Capillary Action
Cohesion/Adhesion
Ion Dipole
H Bond
Dipole- Dipole
Hexane
Dipole- Induced Dipole
Dispersion

8. Chemistry-Van Deemter Equation
H=A+B/u+Cu
Measures efficiency of chromatographic separation
H=Plate Height
u=Velocity of Mobile Phase
A=Eddy Diffusion
B/u=Longitudinal Diffusion
Cu=Mass Transfer

9. Hypothesis and Null Hypothesis
3:1 solution will provide best separation
Isopropyl alcohol-greatest dispersion forces
Lowest polarity
Polar compounds-short distances
Nonpolar compounds-long distances
Null Hypothesis
No relationship or relevance between polarity and separation of pigments

10. Independent and Dependent Variables
Independent Variable
Ratio of 1 M isopropyl alcohol to 1 M water
1:1
3:1
3:2
Dependent Variable
R(f) values of each pigment
no units
Number of pigments

11. Red Leaf Extract-Control
Used as a standard
Helped determine procedure
Data collection

12. Materials Per Trial 3 test tubes 3 mL of cranberry extract
3 strips of chromatography paper
Pipette and pipette pump
18.5 mL 1 M C3H8O
11.5 mL 1 M H2O
Parafilm
Pencil
3 50 mL Erlenmeyer Flasks
Ruler

13. Experimental Setup Preparation of Isopropyl Alcohol/Water Solvent
Preparation of Chromatogram
Data Collection R(f)

14. Determining the Color of Anthocyanin
Done through the Dynamic Model (STELLA)

15. Total Data Collected Cranberry Extract 10 trials 1:1 ratio
Red Leaf Extract (Control)
4 trials 1:1 ratio
2 trials 3:1 ratio
5 trials 3:2 ratio

16. Sample Calculation R(f) Value
Distance traveled by solvent front d(s)=10.7
Distance traveled by compound d(c)=8.0036
R(f)=d(c)/d(s)
=8.0036/10.7
=0.748
Average Pigments per chromatogram
Difference in R(f) values between pigments
Calculated through median and difference functions

17. Data Represented Through Median
Average number of pigments per ratio (median)
Average R(f) value of pigment number (median)

18. Average Number of Pigments per Chromatogram

19. Difference in R(f) Values Between Pigments (Cranberry)

20. Difference in R(f) Values Between Pigments (Red Leaf)

21. Data Analysis Trends 3:2 ratio solvent resolved most pigments
Red leaf extract separated more pigments than cranberry extract
Pigments 1 and 2 have greatest difference in R(f) values
3:2 ratio created largest differences in R(f) values between chloroplast pigments

22. Problems Encountered Recording R(f) values from a chromatogram
Distinguishing between two bands
Determining bands
Determining the furthest extent of the solvent front
Determining when a chromatogram is finished
Remaining solvent

23. How Problems Were Overcome
Define standards for measuring R(f) values
Measure to center of color band
Solvent front-wetness of chromatogram
Bands-variation in color
Excess solvent in tubes-wait ten minutes for notable changes

24. Conclusions 3:2 solvent mixture provided the most effective separation
Largest difference in R(f) values
Most pigments separated on chromatogram
Hypothesis is refuted
Relationship between polarity and chromatographic separation
As the ratio of dispersion forces and dipole- dipole becomes closer, the greater the separation of pigments

25. Future Improvements Create extract More trials
Producing clearer results
UV lamp
Spectrophotometer
Longer chromatograms

26. Questions What are three of the most common pigments in plants?
How does the polarity of the pigments determine how far they will travel up the chromatogram?
By looking at the R(f) values of each pigment, how do you know when effective separation has been achieved?