Observing the Effect of Polarity in the Separation of Pigments Anish Prasanna Jeremy Rubin Aradhana Vyas Block A
Observing the Effect of Polarity in the
Separation of Pigments Anish PrasannaJeremy Rubin
Aradhana VyasBlock 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
o Chlorophyll a and bo Anthocyanino Carotenoids
• Chromatographic Separation
http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chromatography
Basic Chromatography Terms• Mobile Phase
• Stationary Phase
• Band Broadening Theory
• Solvento Solvent Fronto Solution
http://cellbiologyolm.stevegallik.org/aminoacids/page4
Basic Chemistry• Molecular Polarity
o Hydrogen Bondingo Dipole-Dipole Forceso 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+Cuo Measures efficiency of
chromatographic separationo H=Plate Heighto u=Velocity of Mobile Phaseo A=Eddy Diffusiono B/u=Longitudinal Diffusiono Cu=Mass Transfer
http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chromatography
Hypothesis and Null Hypothesis
• Hypothesiso 3:1 solution will provide best
separation Isopropyl alcohol-greatest
dispersion forces Lowest polarity Polar compounds-short distances Nonpolar compounds-long
distances• Null Hypothesis
o 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
watero 1:1o 3:1o 3:2
Dependent Variable• R(f) values of each pigment
o 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
o 10 trials 1:1 ratioo 11 trials 3:1 ratioo 9 trials 3:2 ratio
• Red Leaf Extract (Control)o 4 trials 1:1 ratioo 2 trials 3:1 ratioo 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)=8.0036o R(f)=d(c)/d(s)
=8.0036/10.7 =0.748
• Average Pigments per chromatogram• Difference in R(f) values between
pigments o 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
o 3:2 ratio solvent resolved most pigments
o Red leaf extract separated more pigments than cranberry extract
o Pigments 1 and 2 have greatest difference in R(f) values
o 3:2 ratio created largest differences in R(f) values between chloroplast pigments
Problems Encountered
• Recording R(f) values from a chromatogramo Distinguishing between two bands
o Determining bands
o Determining the furthest extent of the solvent front
• Determining when a chromatogram is finishedo Remaining solvent
How Problems Were Overcome • Define standards for measuring R(f)
valueso Measure to center of color bando Solvent front-wetness of
chromatogramo Bands-variation in color
• Excess solvent in tubes-wait ten minutes for notable changes
Conclusionso 3:2 solvent mixture provided the most
effective separation Largest difference in R(f) values Most pigments separated on
chromatogramo Hypothesis is refutedo 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 resultso UV lampo Spectrophotometer o 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?