Notes

Chromatography:
Chromatography is a lab technique for the separation of a mixture, the process is split into two parts the mobile phase and a continuous stationary stage. The mobile phase can be either a liquid or a gas while the continuous stationary stage is either a solid or a liquid. Chromatography has numerous applications in the industry such as biological and chemical fields. It is used widely in biochemical research for the separation and identification of chemical compounds of biological identification. A specific example is in the petroleum industry the techniques are used to analyze complex mixtures of hydrocarbons. It is used to purify chemicals and to separate compounds. The aim of this investigation is to compare three types of chromatography’s, the paper chromatography of chlorophyll, thin layer chromatography of chlorophyll and the chromatography of amino acids.

Paper Chromatography of chlorophyll:
Introduction:
Chlorophyll is not the only pigment present in leaves, in autumn, chlorophyll breaks down allowing xanthophylls and carotene and anthocyanin to show their colours. This mix of pigments can be separated into bands of colour through the technique of paper chromatography. The word chromatograph, directly translated from its Greek roots means “colour writing”. Using the paper chromatography technique the components of a mixture in a liquid are separated, this takes place by absorption and capillarity. The paper holds the substances by absorption, the capillarity pulls up the substances up the paper at different rates, this causes the pigments to be separated on the paper and show up as coloured streaks with the pattern that it leaves on the paper being called a chromatogram. Paper chromatography is a anylitical method used in the separation of coloured chemicals or substances.

Materials:
Large test tube (24 x 150mmm) to ensure it can hold the filter paper without touching the sides.
Stopper to hold the chromatography paper in test tube.
Pin to hold the chromatography paper in place.
Chromatography paper to show the separation of the various pigments in spinach.
Test tube rack to hold the test tube in place.
Pigment solution, Fresh spinach leaves ground up, killed by rapid immersion in boiling water with three drops of pure propanone, filtered.
Solvent (5cm3) one part 90% propanone and 9 part petroleum ether.

Method:
Draw a line 30 mm from the bottom of the chromatography paper, fold the top of the paper 90o and attach using pin to the stopper ensuring the edges don’t touch the sides and the strip almost touches the bottom.
Remove from boiling tube, using pin as a dropper, place a drop of pigment in the centre of the pencil line.
Wait until pigment dries before placing another small drop on top, repeat until the area is highly pigmented.
Pour 10mm of solvent into test tube, sealed for 10 minutes so the atmosphere inside is saturated with vapour.
Suspend strip in boiling tube making sure it is dripping in but the pigment spot is not immersed.
When the solvent is approximately 20mm from the top of the paper, remove the strip, rule a pencil line to mark solvent front and, dry the paper.
Measure distance from pencil line to the leading edge of each clearly detectable pigment, and work out the Rf value for each.

Hazard: Level of risk: Precaution:
Glassware Low Use with caution, if cut apply pressure to the wound.
Boiling water Medium Use with caution, if burnt immediately put wound under cold water.
Propanone Flammable, irritant , Hazard Highly flammable liquid
Causes serious eye irritation; may cause drowsiness or dizziness; repeated expose may cause skin dryness and cracking.
Petroleum Hazard If contact with skin is made immediately wash with water, if contact is made with eye flood with saline solution and alert medical attention.
Results:

A: 4mm
B: 8mm
C: 10mm

Rf Calculations:
Rf = a/b
A = distance moved by substance from its original position.
B = distance moved by solvent from same position.
A, 4mm / 19mm = 0.21
B, 8mm / 19mm = 0.42
C, 10mm / 19mm = 0.52

Discussion:
Using the sources listed bellow I attempted to identify the pigments using their Rf values from typical and theoretical values, for pigment A the Rf value was 0.21 which lead me to believe it was Neoxanthine due its Rf value being 0.22 however the value is a theoretical value due to the pigment not appearing to the naked eye while in my chromatography there is slight yellow colouring to the pigment which suggest that it could be a Xanthophyll which is a yellow pigment. For pigment B I thought the pigment was chlorophyll b due to it being the closest Rf value to the Rf value that I calculated as well as chlorophyll b being identified as a yellow-green which was similar to the colour of the pigment on my chromatography. For pigment C I identified it as chlorophyll a due to its Rf value being close to the theoretical value as well as the colour of chlorophyll a being a blue-green which I thought I could relate the colour of my pigment to.
I used multiple Rf values because Rf values are different for each solvent yet the general order being similar because more non polar pigments move faster in non polar organic solvents. Furthermore pigments are carried at different rates due to not being equally soluble. The difference in Rf values could be due to this or it could be due to a less accurate method then the other methods I will use later on.
The Rf values that I used were:
1.
Pigments: Theoretical Rf:
Chlorophyll a 0.68
Chlorophyll b 0.54
Chlorophyll b 0.03
B- carotene 0.94
Fucoxanthine 0.51
Lutein 0.43
Neoxanthine 0.22
Violaxanthine 0.08

2.
Pigments: Rf:
Carotene 0.98
Chlorophyll a 0.59
Chlorophyll b 0.42
Pheophytin 0.81
Xanthophyll 1 0.28
Xanophyll 2 0.15

3.
Name: Colour: Rf:
Carotene Yellow 0.95
Phaeophytin Yellow-grey 0.83
Xanthophyll Yellow-brown 0.71
Chlorophyll a Blue-green 0.65
Chlorophyll b Green 0.45







References:
Article title: Chromatography
Website title: En.wikipedia.org
URL: https://en.wikipedia.org/wiki/Chromatography

Website title: Afrox-mozambique.com
URL: http://www.afrox-mozambique.com/en/images/LPG%20%26%20Propane266_122438_tcm444-402892.pdf
2.
Author Homework Help
Article title: Standard Rf Values - Biology (Photosynthesis)
Website title: Physics Forums | Science Articles, Homework Help, Discussion
URL: https://www.physicsforums.com/threads/standard-rf-values-biology-photosynthesis.115321/

Website title: Science.cleapss.org.uk
URL: http://science.cleapss.org.uk/resource/Student-Safety-Sheets-ALL.pdf

Article title: Chromatography | chemistry
Website title: Encyclopedia Britannica
URL: https://www.britannica.com/science/chromatography

Article title: What Is Paper Chromatography and How Does it Work?
Website title: Owlcation
URL: https://owlcation.com/stem/What-is-Paper-Chromatography-and-How-does-it-Work

Article title: Chromatography Plant Pigments - BIOLOGY JUNCTION
Website title: BIOLOGY JUNCTION
URL: https://www.biologyjunction.com/chromatography_plant_pigments.htm
1.
Article title: Why Do Different Pigments Have Different Rf Values?
Website title: Reference
URL: https://www.reference.com/science/different-pigments-different-rf-values-9a50be9b7454e044
3.
Author M. B. V Roberts
Year published: 1976
Book title: Biology: a functional approach
City: Walton on Thames
Publisher: Nelson (T.)