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

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

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

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

Paper Chromatography Introduction Chloroplast Pigments Chlorophyll a and b Anthocyanin Carotenoids Chromatographic Separation http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chromatography

Basic Chromatography Terms Mobile Phase Stationary Phase Band Broadening Theory Solvent Solvent Front Solution http://cellbiologyolm.stevegallik.org/aminoacids/page4

Basic Chemistry Molecular Polarity Hydrogen Bonding Dipole-Dipole Forces Dispersion Forces Solubility Capillary Action Cohesion/Adhesion Ion Dipole H Bond Dipole- Dipole http://www.science.uwaterloo.ca/~cchieh/cact/c123/intermol.html Hexane Dipole- Induced Dipole Dispersion

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 http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chromatography

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

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

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

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

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

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

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

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

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

Average Number of Pigments per Chromatogram

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

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

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

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

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

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

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

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?