Period+2+-+CS+-+Final+Report

=Chromatographic Separation=
 * Due 01 December, 2008**

Back to Period 2 - CS

Video Footage of the Experiment
media type="youtube" key="CD4hQAVxPQI" width="425" height="350"

Introduction
Chromatography is the term used to describe the various different types of laboratory processes that can be used to separate mixtures. All types of chromatography involve separating a mixture between a mobile phase and a stationary phase. The mixture is on the stationary phase (a solid or a liquid) and the mobile phase (fluid) is passed through it. Chromatography can be preparative or analytical. The purpose of Preparative chromatography is to separate the components to be used for other purposes. Analytical chromatography aims to measure the relative proportion of the analyte in the mixture.

The different types of chromatography can be categorized into five different sections: **Adsorption, Partition, Ion Exchange, Molecular Exclusion, and Affinity.** Here, we will focus on Adsorption.

Adsorption Chromatography is the process where a mobile gas or liquid solute accumulates onto the surface of a stationary solid, forming a thin layer of molecules. Unlike other types of chromatography, adsorption is usually a liquid-on-solid extraction. First, the sample that is to be tested is placed on the solid, called the adsorbent. The adsorbent is a solid that is capable of binding the components on it into a mixture. Next, the solid is dipped into a solvent (such as H2O). As the solvent travels up the solid, it meets and dissolves the sample, creating the adsorbate. Then, it will continue up the paper as a solvent solute sample. Different compounds in the sample will result in the solute traveling at different rates, depending on the solubility of the solvent, and the attraction between the solvent and the solid. With relation to chemical dyes, the compounds that make up the dyes will behave differently, which would easily distinguish them between each other. In this experiment, adsorption is used to separate the different compounds in coloured pigments. The adsorbent is chromatography paper, the solute is the coloured pigment in various Sharpie pens, and the solvent is the Isopropyl Alcohol. Once the solvent reaches the solute, the adsorbate is formed. However, there are many different compounds in each coloured pigment. If one of the compounds has low solubility, that specific adsorbate will travel faster up the chromatography paper. This is because the low solubility results in a longer time as a liquid solution, which in turn results in a longer time travelling upwards via capillary action. Consequently, a higher solubility equals a shorter time going upwards, as the adsorbate will be in its stationary phase for a longer time before travelling upwards. This would, in turn, show the dispersion of different colours because of their various solubilities.

This experiment uses the analytical technique known as Paper Chromatography. Paper chromatography utilizes paper as the stationary phase and a liquid which doesn’t react with paper as the mobile phase. It is used primarily to separate and identify colored mixtures. For more complicated substances with similar compositions such as amino acids, two-way paper chromatography is recommended which uses two solvents.

The success of this type of chromatography is rooted in a solvents ability to move up paper by capillary action which is a result of the solvent molecules attraction to the paper and one to one another. The solvent rises up the paper, mixes with the sample mixture and continues to travel upwards. Due to differences in their attraction to the fibers in the paper and the differences in solubility in the solvent, different compounds travel upwards at different rates.

Rf (retention factor) values are used to determine particular components of the mixture. As long as the type of paper, exact composition of the solvent and other aspects of the experiment are consistent, the Rf value for a particular sample will always remain the same.

Rf is found using the following equation:

__Distance travelled by sample__ Distance travelled by solvent

The experiment further described below will demonstrate how paper chromatography can be utilized to separate inks or pigments.

Materials

 * Glass Jars
 * Chromatography Paper
 * Pencils
 * Rubbing alcohol (70% Isopropyl Alcohol)
 * Sharpie Pens of various colours
 * Measuring cup
 * Ruler
 * Scissors
 * Tape
 * Safety Goggles

Procedure

 * 1) Use the scissors and ruler to cut equally long strips of Chromatography Paper. The number of strips should be equivalent to the number of jars. Then, in pencil, draw a horizontal line 1 cm above the bottom edge of each strip.
 * 2) Using each Sharpie pen, place a spot along the line of the Chromatography Paper. Place no more than 3 spots on each strip. Write down all the colours and where each colour's spot is located.
 * 3) Tape each strip onto a different pencil, and place the pencil onto the jar. Make sure that the strip of Chromatography Paper is touching the Isopropyl Alcohol inside, but does not pass the pencil line.
 * 4) Measure approximately 15 mL of Isopropyl Alcohol and pour it in each jar.
 * 5) Let the strips develop until the ascending solution is approximately 2 cm from the top of the paper. Then remove the strips and let them air dry.
 * 6) Record your observations and determine the retention factor.

Qualitative: Colour Observations

 * **Spot Colour** || **Adsorbate Colour** ||
 * Black || Blue, Orange, Red ||
 * Gray || Purple ||
 * Blue || Blue ||
 * Green || Green, Yellow ||
 * Light Green || Green, Yellow ||
 * Orange || Orange, Red ||
 * Burgundy || Burgundy ||
 * Green (2) || Green, Yellow ||
 * Orange (2) || Orange, Red ||
 * Burgundy (2) || Burgundy ||

Quantitative: Data Table
Red || 4.3cm, 1.6cm, 3cm || 4.3cm || 1, 0.3271, 0.6977 || The (2) indicates a second trial
 * **Spot Colour** || **Adsorbate Colour** || **Distances Travelled (Respective)** || **Distance Travelled by Solvent** || **Retention Factor (Respective)** ||
 * Black || Blue, Orange,
 * Gray || Purple || 4.3cm || 4.3cm || 1 ||
 * Blue || Blue || 3.9cm || 3.9cm || 1 ||
 * Green || Green, Yellow || 4.1cm, 4.4cm || 4.4cm || 0.9318, 1 ||
 * Light Green || Green, Yellow || 4cm, 4.4cm || 4.4cm || 0.9090, 1 ||
 * Orange || Orange, Red || 3.1cm, 4cm || 4cm || 0.7750, 1 ||
 * Burgundy || Burgundy || 4cm || 4cm || 1 ||
 * Green (2) || Green, Yellow || 3.8cm, 4.2cm || 4.2cm || 0.9048, 1 ||
 * Orange (2) || Orange, Red || 3.2cm, 4.2cm || 4.2cm || 0.7619, 1 ||
 * Burgundy (2) || Burgundy || 4cm || 4cm || 1 ||

Pictures:
2: Crayola Grey || 1: Crayola Blue 2: Sharpie Blue || 1: Sharpie Green 2: Sharpie Light Green || 2: Sharpie Orange || 1: Sharpie Orange 2,3: Sharpie Burgundy (x2) ||  ||
 * [[image:BLK_GRA.JPG width="215" height="169"]] || [[image:BLU_BLU.JPG width="219" height="168" align="center"]] || [[image:GRN_LGR.JPG width="208" height="168" align="center"]] ||
 * 1: Sharpie Black
 * [[image:GRN_OJ.JPG width="214" height="164" align="center"]] || [[image:OJ_RED.JPG width="198" height="166" align="center"]] ||  ||
 * 1: Sharpie Green

Calculations
Retention factors (Rf) for each sample were calculated for this experiment. As mentioned earlier, Rf is found using the following equation:

__Distance travelled by sample__ Distance travelled by solvent

We performed this calculation on each sample. Please see the data table for the results.

Discussion
First, all of the permanent markers that were used were polar. This is true because if they were not polar, they would not move from the 1 cm pencil line. It was found that most, if not all permanent markers (most noticeably, Sharpies) have ink made of different alcohols, including diacetone and butyl alcohol.

It was discovered that the inks that were tested twice had very close retention factors. The miniscule difference would have been caused by a small difference in the amount of ink used in each chromatogram, which would have altered the amount of distance it travelled. However, both burgundy samples produced the same retention factors. This experiment demonstrates that no matter how many times a mixture is separated under the same conditions, the retention factor will be the same.

Chromatography has many practical uses. The many types of chromatography is used in science, medicine, and law enforcement. Paper chromatography, for instance, can be used to identify unknown organic and inorganic compounds. It is also used in crime scene investigations, DNA and RNA sequencing, and others. Any kind of work that involves separating a solution requires some form of chromatography.

Sources of Experimental Error
Errors could have occured in a number of places during this experiment. First of all, we were uncertain as to whether or not the chromatography paper was in good condition. We could have accidentally damaged it when we were taking it home. Secondly, the measurements made were not 100% precise; we used a ruler, not a special measuring tool with extreme precision. Although every effort was made to make the size of the dots the same, it was an excruciatingly difficult task, and we could not be certain if their sizes are 100% the same. All of these sources of error could have affected, albeit very slightly, the outcome of our experiment.

Conclusion
This experiment focused on the separation of mixtures through adsorption and paper chromatography. Relative strengths of the forces of intermolecular attraction between substances could be determined by the speed of the separation (the faster the line moves in the chromatography paper, the weaker the forces of attraction). This is because a stronger intermolecular force is harder to break, and therefore it will spend a longer time in the stationary phase blending before moving upwards.

Retention factors (Rf) were also measured for each substance used in this experiment. Rf can help identify an unknown compound. If an unknown compound and a known compound have the same retention factor, they may be the same substance, but it may not always be the case. However, if they do not have the same retention factor, then they are certainly not the same compound. This experiment showed how well the markers used attract to the surface of paper. Furthermore, having a retention factor (for every solute tested) means that all the solute tested were polar. This is because solutes that are not polar would not move at all; they will stay as a dot on the line. The nonpolar solute will not be able to dissolve in isopropyl alcohol (polar), since it has a neutral "charge" and has no polarity. 

Suggestions For Modifications
After a thorough review of our experiment, there are several possible modifications that could have been done to make the experiment better. First, different types of solutes may produce different colours and Rf values. Some examples of different solutes could include food colouring or a homemade mixture. A variety of different types of solvents could be used as well. Different concentrations of isopropyl alcohol, other than 70%, would produce different values for Rf. In addition, by testing solvents with known chemical formulas, it would be easier to determine the polarity of the specific solvent, as well a relationship between polarity, retention factor, and the different colours that are formed. Also, it would be helpful if we could record a video or time how fast the solution was travelling up the chromatography paper for each specific colour. Then, it would allow more relationships to be made (with respect to time) such as a solution's speed.

Safety Concerns
It was at times difficult to pour the solvent (70% Isopropyl Alcohol) out without spilling or splashing. This is important as the Alcohol can permanently damage many materials and other objects. Furthermore, do not consume the alcohol. Due to its high concentration, the alcohol is highly toxic and is very harmful to one's body. Also, doing the experiment in a well ventilated area is recommended, as the odour of rubbing alcohol may be irritating.

Refering to the permanent markers, do not play around with these markers, as they can stain clothing and other items. It would also be wasteful and it can possibly alter results in the experiment if the ink starts to run out.