Chromatorgraphy

Experiment 3:

Paper Chromatography

27th of January, 2016

Done by: Labib

Group member(s): Barsha

Aim/Purpose: 

To achieve the best possible separation of spinach pigments by using paper chromatography were 4 different solvents would be used and were the pigments would be separated and identified based upon how far they travel up the paper.

 Variables:

Independent Variables:

• Type of substances (spinach) used
• Type of materials and equipment used
• Time

Dependent variables:

• Rf (retention factor) value
• Solvent front
• Pigment distance from origin

Controlled variables:

• Room temperature
• Amount of spinach and solvent used
• Type of materials and equipment used

Hypothesis/Prediction:

My prediction in this experiment is that, since we are using four different solvents in order to separate the spinach pigments, the distance that each of these pigments travel would be different in the case of each solvent. This is due to the differences in the attraction between the pigments and the different solvents. In chromatography, the pigments are separated due to differences in solubility with selected solvents. Hence, the pigments would be carried at different rates and reach different distances on the paper since they are not equally soluble.

Background information:

Chromatography is a method of chemical analysis used to separate and then identify the components of mixtures. It is a process that separates molecules because of the different solubility and attractions of the molecules in a selected solvent. The chemical basis of chromatography is that different components will adsorb onto a surface and desorb into a solvent at different rates. There are 3 main parts to any analysis involving chromatography which are:

• Mixture: contains components/compounds/elements to be separated
• Stationary phase: mixture is placed onto the stationary phase. Components in the mixture are attracted to the stationary phase in by varying intermolecular forces
• Mobile phase: a specially selected solvent that will travel through the stationary phase. As it travels, depending on the solubility and intermolecular forces, the components of the mixture will either be attracted to the solvent and move up the stationary phase or will not be attracted at all.

With contrast to that paper chromatography is basically another type of chromatography were a special filter paper is used to separate and identify different components within a mixture most notably, the different pigments within plant extracts.  In this technique, the mixture contain the pigments to be separated is first applied as a spot or a line to a thin strip of filter paper about 1.5 cm from the bottom edge of the paper. The paper is then placed in a container with the tip of the paper touching the solvent. The solvent is then absorbed by the paper and moves up due to capillary action. As the solvent crosses the area containing the plant extract, the pigments dissolve in and start moving with the solvent. However depending on the intermolecular forces and other factors such as solubility, molecular weight, geometry etc. the pigments are carried at different rates. For example, pigments that are polar are most likely to be soluble within the solvent and hence move up the paper at a faster rate. Components that have a stronger attraction with the stationary phase (paper) than the solvent will move more slowly than those that are polar. The polarity of a solvent is also taken into account in this case. Solvents that are totally polar such as water are not used in chromatography due to the fact that it has a high polarity index. A polarity index is a set of values which represents the polarization for certain solvents.  The distance the pigment travels is unique to each pigment and is used for identification.  Like this, the pigments get separated from the plant extract forming different colors and hence, producing a chromatogram.

Materials and Equipment:

• Test tube (4)
• Test tube rack (1)
• Spinach leaves (2 to 3 pieces)
• 1 A4 sized chromatography paper (coffee filter, filter paper etc.) (4 strips)
• Mortar and pastel
• Solvents used (each 1mL):

• N-propyl Alcohol (Solvent D)
• Vinegar ( Solvent B)
• Ethanol (Solvent C)
• Methanol (Solvent A)

• Pipette(s) (3)
• Tweezer(s) (1)
• Scissor(s) (1)
• Ruler(s) (1)
• Pencil(s) (1)
• Lab safety equipment (Gloves, goggles, lab apron etc.)

Procedure:

1. An A-4 sized coffee filter was taken which was then used to cut 4 strips of paper used throughout the experiment
2. 4 strips of chromatograph paper was drawn using a ruler and pencil, each with a length of 11.5 cm and width of 1 cm was cut with scissors in order to match the length and width of the test tubes
3. A line was then drawn on all four strips 2cm from the bottom of each strip horizontally.
4. The bottom of each strip was then cut in order to form a pointed end

[pic 1]

1. Using a mortar and a pastel, pieces of spinach leaves were crushed in methanol in order to form a spinach-methanol solution.
2. A pipette was then used to collect 1ml of spinach-methanol solution and put over the line on the strip. This process was repeated for the remaining 3 strips with 3 different pipettes[pic 2]
3. The strips were then taken to dry for 20 minutes
4. 4 test tubes were taken, each filled with 1mL of each solvent which was then sealed with a cork and kept in the test tube rack
5. After 20 minutes with the help of a tweezer, all four strips were carefully lowered into the test tubes with the cork secured on top. It was made sure that the solvent touched the pointed end of the paper but not the green line

1. Solvent movement and band separation was observed for 20 minutes
2.  When the pigments had separated into distinct bands (the solvent has moved approximately have the distance of the paper), all 4 of the strips were then taken out of the test tube
3. All the strips were allowed to dry for 5 minutes
4. The Rf value was then calculated for all of the pigments by dividing the distance the pigment travels by the distance if the solvent front
5. All data that was observed throughout the experiment was then recorded on a data table

Results/Observation data:

The following is all the data that was obtained during the course of our experiment:

At the end of our experiment, all four of the strips (each experimented with a different solvent) looked like this:

[pic 3]

Rf Value: In paper chromatography, the distance the mixture has moved from the origin compared to the distance the solvent has moved from the origin is known as the Rf value. The formula for calculating the Rf value is-[pic 4]