Observing the Effect of Polarity in the Separation of

Pigments Anish PrasannaJeremy RubinAradhana 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

http://www.science.uwaterloo.ca/~cchieh/cact/c123/intermol.html

Ion Dipole H Bond Dipole- Dipole

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

● 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○ 11 trials 3:1 ratio○ 9 trials 3:2 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?