P1+CS+Final

toc =**P1 Chromatographic Separation Final**=
 * Our Browsers had some problems with the Wikispace - please excuse the formatting issues with font/subscript BUT IT WORKED WITH GOOGLE CHROME

**Introduction**
Chromatography is the diverse group of physical methods for separating complex mixtures into the chemicals that they are composed of. It involves the mixture being on a ‘stationery phase” (solid, liquid, or gel form) while a fluid in the “mobile phase” travels in a definite direction through it. The fluid or solvent in the mobile phase separates the various substances of a mixture, allowing them to be studied. There are two reasons for chromatographic experiments – Preparative and Analytical. The purpose of Preparative Chromatography is to separate the substances for further use, while Analytical Chromatography aims to measure the relative proportions of substances in a mixture. Preparative Chromatography is the more easily studied of the two.

The first chromatograph was invented by Russian botanist Mikhail Semenovich Tsvett. Tsvett was looking for a method of separating a mixture of tints which are chemically very similar to each other. To isolate different types of chlorophyll, he trickled a mixture of dissolved pigments through a glass tube packed with calcium carbonate powder. As the solution washed downward, each pigment stuck to the powder with a different degree of strength, creating a series of coloured bands. Each band of color represented a different substance.

There are five different types of Chromatography, namely: Partition, Ion Exchange, Molecular Exclusion, Affinity, and Adsorption. We will examine Adsorption Chromatography in depth later.

Partition chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. The solute equilibrates between the mobile phase and the stationary liquid. In Ion-Exchange chromatography, the use of a resin is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. Molecular Exclusion Chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size. The pores are normally small and exclude the larger solute molecules, but allow smaller molecules to enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass through the column at a faster rate than the smaller ones.

Affinity chromatography utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. When solutes containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase. This protein is later extracted by changing the ionic strength or pH.

Metal Chelation Chromatography or Immobilized Metal Ion Affinity Chromatography, a subtype of both Ion Exchange and Affinity Chromatography is used to separate metal ions. In this type, cations react with imidazols (histidine residues) in proteins of carboxylate resins. The isolation will occur if there is an excess of free imidazol. This type of Chromatography is far beyond our scope and ability of classroom experimentation.

**Adsorption Chromatography**
Adsorption occurs when a fluid solute accumulates on the surface of a solid or liquid, forming a thin film of molecules or atoms. It is different from absorption, in which a substance diffuses into a liquid or solid to form a solution. Adsorption Chromatography takes advantage of the fact that different components of a mixture interact differently with the two phases. Some substances in the mixture will be more strongly adsorbed to the stationery phase, while others will be more soluble in the mobile phase. As the mobile phase moves through the stationery phase, the substances that are easily adsorbed will lag behind the other substances, creating a separation. Thus, when separating chemical dyes through Chromatography, the substances within each dye will ‘lag’ at different rates, causing a separation that is easily seen.

**Paper Chromatography**
Paper Chromatography is a type of Adsorption Chromatography in which the stationery phase used is paper. It was invented by two British biochemists, Archer John Porter Martin and Richard Laurence Millington Synge. In 1941, they began working on proteins, which are made up of chains of amino acids. They were trying to characterize a particular protein by determining the precise numbers of each amino acid present. Since Amino acids are similar to each other, separating proteins proved difficult. It was discovered that a strip of porous filter paper could substitute for the column of absorbing powder used by Tsvett. The development of paper chromatography to solve their problem was a success.

In paper chromatography, a drop of the mixture to be separated is placed on the paper, and then one edge is dipped into the fluid (in this case liquid) phase. Through capillary action, the liquid moves up the paper. Once adsorption occurs and the paper dries, a spray-on reagent reveals the change in color based on differences in solubility and adsorption. Paper Chromatography’s main purpose is to separate and identify mixtures that are coloured, such as pigments, dyes, or inks.

A useful type of paper chromatography is Two-way Paper Chromatography, which employs two different solvents (mobile phases) and rotating the paper 90 degrees. Two-way Paper Chromatography helps separate mixtures whose constituents are all similar compounds, such as amino acids.

In industrial settings, Paper Chromatography has been replaced by Thin Layer Chromatography, which uses a gel instead of paper for the stationery phase. The gel provides more accurate results in situations where specifics are necessary.

Rf is defined as the:
 * ratio of fronts;
 * rate of flow; or
 * retention factor.

In any case, it is the value of the "distance travelled by a component" in a chromatographic adsorption experiment divided by the "distance travelled by the mobile phase".

If the same mobile and stationery phases are used, the Rf values are the same for a particular sample in any mixture. These values can be used to determine the specific substances present. (If the Rf value and colour observed in Paper Chromatography of two substances are the same, it is likely that they are the same substance.)

This experiment will examine how to separate mixtures through adsorption and Paper Chromatography, and determine the substances present.

**Materials**

 * Glass Container with Rack
 * Toothpicks
 * 6 Pigments
 * Food Coloring (2 Mixtures: Brown = Red + Yellow + Blue; and Green = Yellow + Blue)
 * 100% Pure Spinach Extract, 100% Pure Pomegranate Extract
 * 4 Strips of Chromatography Paper of Equal Length
 * 70% Isopropyl Rubbing Alcohol (Solvent/Adsorbent)
 * Funnel, etc.for Safe Pouring
 * Pencil
 * Ruler
 * Observation Sheet
 * Safety Goggles

**Procedure**

 * I do remember you saying something about how the procedure shouldn't be numbered, it's merely a summary of what was done. I couldn't remember so the first version of our procedure is numbered, the second is the exact same but in paragraph form. Thanks.


 * Revised Procedure** (Various Changes were made to the original draft according to conditions and availability of materials)**:**

Numbered:
 * 1) A pencil was used to mark a line on each strip of Chromatograpy Paper, approximately 1 cm from the edge.
 * 2) Toothpicks were used to apply a small spot of each pigment to a location just above each aforementioned line - one pigment per line.
 * 3) Each strip of Chromatography Paper was labelled using a pencil, based on the type of pigment that was applied to it. (i.e. Food Coloring Mixture, Spinach Juice, etc.)
 * 4) Each strip was hung off of the rack at equal lengths so that the bottom edges lined up.
 * 5) The rack was then placed in the Glass Container and the container was filled with enough solvent (70% Isopropyl Alcohol) so that a level just below the spots was reached.
 * 6) The rack was removed from the container once the solvent in the "Chromatographic Chamber" had travelled to the top of each strip of chromatograpy paper.
 * 7) The final position of the solvent on the strips was then marked with a pencil. (This value was common for all strips.)
 * 8) The final position of each initial pigment was also marked with a pencil. (If the initial pigment separated into multiple colours, each colour's final position was marked.)
 * 9) The "distance travelled by the mobile phase" was measured (in mm) using the ruler and recorded on the observation sheet. (This distance was from the top of the initial spots to the final position of the solvent.)
 * 10) The "distance traveled by each colour" was measured (in mm) using the ruler and recorded on the observation sheet. (This distance was from the top of the initial spots to the final position each colour.)

Paragraph:

A pencil was used to mark a line on each strip of Chromatograpy Paper, approximately 1 cm from the edge. Toothpicks were used to apply a small spot of each pigment to a location just above each aforementioned line - one pigment per line. Each strip of Chromatography Paper was labelled using a pencil, based on the type of pigment that was applied to it. (i.e. Food Coloring Mixture, Spinach Juice, etc.) Each strip was hung off of the rack at equal lengths so that the bottom edges lined up. The rack was then placed in the Glass Container and the container was filled with enough solvent (70% Isopropyl Alcohol) so that a level just below the spots was reached. The rack was removed from the container once the solvent in the "Chromatographic Chamber" had travelled to the top of each strip of chromatograpy paper. The final position of the solvent on the strips was then marked with a pencil. (This value was common for all strips.) The final position of each initial pigment was also marked with a pencil. (If the initial pigment separated into multiple colours, each colour's final position was marked.) The "distance travelled by the mobile phase" was measured (in mm) using the ruler and recorded on the observation sheet. (This distance was from the top of the initial spots to the final position of the solvent.) The "distance travelled by each colour" was measured (in mm) using the ruler and recorded on the observation sheet. (This distance was from the top of the initial spots to the final position each colour.

Data Tables
Data Table of distance travelled in cm by each component:
 * ** Component ** || ** Distance Travelled (cm) ** ||
 * Adsorbent/Mobile Phase (70% Isopropyl Alcohol) || 7.25 ||
 * Red Food Coloring (In Mixture) || 7 ||
 * Yellow Food Coloring (In Mixture) || 6.8 ||
 * Blue Food Coloring (In Mixture) || 7.1 ||
 * Green Food Coloring (Yellow Component) || 2.3 ||
 * Green Food Coloring (Blue Component) || 7.1 ||
 * Spinach Juice (Yellow Component 1) || 7 ||
 * Spinach Juice (Yellow Component 2) || 4 ||
 * Spinach Juice (Green Component) || 3.5 ||
 * Pomegranate Juice (Orchid Purple Component) || 6.1 ||
 * Pomegranate Juice (Light Salmon Pink Component) || 4.6 ||

**Pictures** The following pictures show the process of adsorption through the progression of

1) The Mixture involving Red, Yellow, and Blue Food Colouring Pigments: 2) The Spinach and Pomegrante and Blue Pigments:

Calculations
Rf was calculated using the following formula:

Rf = (distance travelled by colour) / (distance travelled by the mobile phase)

Since the distance travelled by the mobile phase was a constant 7.25 cm, this formula can be modified to become: Rf = (distance travelled by colour in cm) / 7.25 cm Using this method, the values were calculated for each component.

Table of Values of Rf:
 * ** Component ** || **Rf** ||
 * Red Food Coloring (In Mixture) || 0.97 ||
 * Yellow Food Coloring (In Mixture) || 0.94 ||
 * Blue Food Coloring (In Mixture) || 0.98 ||
 * Green Food Coloring (Yellow Component) || 0.32 ||
 * Green Food Coloring (Blue Component) || 0.98 ||
 * Spinach Juice (Yellow Component 1) || 0.97 ||
 * Spinach Juice (Yellow Component 2) || 0.55 ||
 * Spinach Juice (Green Component) || 0.45 ||
 * Pomegranate Juice (Orchid Purple Component) || 0.84 ||
 * Pomegranate Juice (Light Salmon Pink Component) || 0.63 ||

**Conclusion**
This experiment examined the separation of mixtures using adsorption and paper chromatography. It was observed that some components in mixtures move more quickly than others. This is due to forces of intermolecular attraction, namely Hydrogen Bonds, Dipole-Dipole attraction and London’s Dispersion Forces. When the components of the mixture are separated via chromatography, they will move much faster if the intermolecular bonds are weak, hence they will move slower if their intermolecular bonds are strong. If the component moves slowly it is probable that it forms hydrogen bonds as they are the strongest type of intermolecular attraction. Also, if two components move through the chromatogram at relatively the same speed, it is likely that London’s Dispersion Forces are the only difference between the chemical structures of the two substances.

The real source of success for the chromatogram is adsorption. Adsorption is similar to solubility in the sense that like dissolves like, in terms of polarity. A solute of low polarity will be adsorbed more strongly to an adsorbent of low polarity than one of high polarity. Thus, when using an adsorbent of low polarity such as the isopropyl alcohol used, the components of low polarity were observed to travel further than others. In this way, polarity is a major factor when determining the components of a mixture that has passed through a chromatogram.

The intermolecular forces of attraction cause variant values for the Retention Factor / Rate of Flow (Rf) of each pigment. This allows the identification of a component based on its position after adsorption if its molecular structure is previously known. If the molecular formula is unknown, a crude form can be determined based on relative Rf values. Chromatography is an accurate way of identifying components in a mixture. Chromatography has many practical applications. It is used in toxicology, the study of sports medicine and can be used to determine if a reaction is complete. Paper chromatography in particular is a vital process used every day. Its many uses include: separating amino acids and anions, RNA fingerprinting, separating and testing histamines and antibiotics. As observed, it can also be used to determine the composition of plants and vegetables such as spinach. Its uses extend to any field in which separation of various substances is necessary. Unfortunately, the substances obtained were not in a usable form once separated. This was found to be one of the hindrances of paper chromatography. Also, as observed with the pomegranate juice, if the compounds in a mixture have a similar colour it can be very difficult to distinguish them from one another.

Discussion
In this experiment, the process of the adsorption separated the various components of mixtures based on their molecular structures. Mainly, a solute that had a similar polarity (low) to the adsorbent of ispropyl alcohol would be adsorbed more and carried further, giving a high Rf value. Solutes with hgih polarity would have low Rf values. Hydrogen bonding capability can also contribute to adsorption. Components with recognizable colours were used to demonstrate this clearly.

For example, this experiment included the separation of a pigment that included coloured dyes of red, yellow and blue. These dyes each have separate molecular compositions and so varied Rf values. It was observed that the blue red and yellow dyes had Rf values of 0.97, 0.94, and 0.98 respectively. The molecular structures of the components are as follows: Red - C 18 H 14 N 2 Na 2 O 8 S 2; Yellow - C 16 H 10 Na 2 O 7 S 2 N 2; Blue - C 16 H 10 N 2 O 2. The long hydrocarbon chain in each is responsible for low polarity, high adsorption, and high Rf values. Also, each component's structure allows for Hydrogen bonding and so the substances are more easily adsorbed.

The green coloured dye separated into two components in the experiment; a yellow and a blue component. Later analysis concluded that the yellow dye had a molecular structure of C 16 H 9 N 4 Na 3 O 9 S 2, with a high polarity and thus was only slightly adsorbed giving a low Rf value. The blue component had the same molecular structure as that in the previous mixture, and so the Rf value remained constant.

One of the pigments that were used was the distinctive green pigment of spinach extract. It was observed that the spinach juice separated into 3 distinct colours. There were two different shades of yellow and one shade of green. The green shade was determined to be Chlorophyll B (C 55 H 70 O 6 N 4 Mg), which is commonly found in spinach leaves. It has the ability to form hydrogen bonds, which would explain the low Rf value of 0.45. Also, its structure is polar and so would not be adsorbed much by the adsorbent. Carotenes and Xanthophylls are likely the causes of the yellow pigmentations as they are also both found in spinach. Carotenes (C 40 H 56 ) were believed to be the source fo the yellow pigment with and Rf value of 0.97 as its molecular structure of a long hydrocarbon chain has a very low polarity. Xanthophylls (C 40 H 56 O 2 ) are likely the source of the other yellow component that had an Rf value of 0.55. Xanthophylls are very similar in molecular structure to Carotenes but have an added O 2 molecule which greatly increases polarity.

The experiment also included the separation of pomegranate extract. Two separate spots of concentration were found with Rfs of 0.84 and 0.63. It is a possibility that the substance with the Rf of 0.84 could form hydrogen bonds but the substance with an Rf of 0.63 could not. Another possibility is a difference in polarity or a strong difference in London’s Dispersion Forces that caused the discrepancy. It was difficult to distinguish which components of the pomegranate juice were observed due to similarities both in colour and in the Rf values. This is one example of Paper Chromatography’s limitations.

Suggestions for Modifications
As with all studies in science, modifications can be made to improve the quality of the results obtained. Firstly, a more diverse selection of pigments (including naturally occurring and non-naturally occurring ones) would have provided definite results that could be applied to a broader range of studies. Secondly, the experiment could be repeated using various adsorbents, such as distilled water, alcohols with different concentrations, etc. The knowledge of whether the solvent affects Rf values would be especially beneficial to areas of study such as RNA separation where exact data is neccessary. It would also be interesting to note if the chemical structure of these solvents impacted the values, and whether or not solutes react differently to solvents with various intermolecular attraction capabilities, particularly polarity. This would be similar to reverse chromtography, in which an adsorbent of high polarity is used and inverse results are expected. An attempt at 2-Dimensional Paper Chromatography would also be beneficial to the data obtained. In 2-Dimensional Chromatography, the process of adsorption is completed once in the normal way, followed by a second process after the chromatography surface has been rotated 90 degrees. A different adsorbent is also used for the second process, causing more definite separations of components. This technique would be particularly helpful in the case of the pomegrante extract which contained 2 substances off very similar colour.

Sources of Experimental Error
Experimental Errors may have occurred at various points during the experiment. The most probable source of error was in the size of the dots of each pigment. Although attempts were made to be as accurate as possible in applying the pigments, slight discrepancies may have affected the resultung Rf values. Also, in keeping with accuracy, the length that the strips were lowered into the alcohol should have been measured to provide more precise information. A difference in this value may have swayed the values for Rf.

**Safety Concerns**
Three main safety concerns were addressed:
 * 1) Safety goggles must be worn at all times.
 * 2) Any contact with the alcohol must be avoided.
 * 3) The chromatogram must be kept in a well ventilated area as alcohol is very flammable and has a strong odour.