P1+CS+Draft

=DO NOT EDIT FURTHER= = = =P1 Chromatographic Separation Draft=

Note:

~Indicates what is due Monday but not to be filled out
 * Indicates what is due Monday

*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 traveled by a component" in a chromatographic adsorption experiment divided by the "distance traveled 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.)

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

*Materials

 * 8" Aluminum pie plate
 * Toothpicks
 * 8 Pigments [Food Coloring (4), Fine Tip Ink Pens (4)]
 * Paper Coffee Filters (Chromatography Paper if available)
 * Solvent - 70% Isopropyl Rubbing Alcohol
 * 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.

Numbered:


 * 1) Eight evenly spaced dots were marked around the coffee filter with a pencil, approximately 1 cm from the edge.
 * 2) Toothpicks were used to apply a small spot of each food coloring to a location just above each aforementioned line on one side of the filter - one coloring per line.
 * 3) The Ink Pens were applied to the 4 remaining lines.
 * 4) Each spot was allowed to dry and then more of the same colour was applied to the spot.
 * 5) The top of each spot was marked with a pencil. Below each spot, the pencil was used to label the type of ink or coloring. (i.e. Green Pen – G.P, Red Food Coloring – R.C)
 * 6) The coffee filter was placed upside down in the aluminum pie plate and the pie plate was filled with enough solvent (Rubbing Alcohol) so that a level just below the spots was reached.
 * 7) The coffee filter was removed from the pie plate once the solvent in the "Chromatographic Chamber" had traveled to the top of the coffee filter.
 * 8) The final position of the solvent on the filter was then marked with a pencil.
 * 9) The final position of each initial color was also marked with a pencil. (If the initial colour separated into multiple colours, each colour's final position was marked.)
 * 10) The "distance traveled 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.)
 * 11) 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:

Eight evenly spaced dots were marked around the coffee filter with a pencil, approximately 1 cm from the edge. Toothpicks were used to apply a small spot of each food coloring to a location just above each aforementioned line on one side of the filter - one coloring per line. The Ink Pens were applied to the 4 remaining lines. Each spot was allowed to dry and then more of the same colour was applied to the spot. The top of each spot was marked with a pencil. Below each spot, the pencil was used to label the type of ink or coloring. (i.e. Green Pen – G.P, Red Food Coloring – R.C) The coffee filter was placed upside down in the aluminum pie plate and the pie plate was filled with enough solvent (Rubbing Alcohol) so that a level just below the spots was reached. The coffee filter was removed from the pie plate once the solvent in the "Chromatographic Chamber" had traveled to the top of the coffee filter. The final position of the solvent on the filter was then marked with a pencil. The final position of each initial color was also marked with a pencil. (If the initial colour separated into multiple colours, each colour's final position was marked.) The "distance traveled 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 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.)

~Data Tables
Sample Data Table of Distance travelled by each component:


 * ~ Component ||~ Distance Traveled (cm) ||
 * Adsorbent ||  ||
 * Red Food Coloring ||  ||
 * Yellow Food Coloring ||  ||
 * Green Food Coloring ||  ||
 * Blue Food Coloring ||  ||
 * Red Marker ||  ||
 * Yellow Marker ||  ||
 * Green Marker ||  ||
 * Blue Marker ||  ||

~Calculations
Calculate Rf using:

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

Display values in table:  
 * ~ Component ||~ Rf ||
 * Adsorbent ||  ||
 * Red Food Coloring ||  ||
 * Yellow Food Coloring ||  ||
 * Green Food Coloring ||  ||
 * Blue Food Coloring ||  ||
 * Red Marker ||  ||
 * Yellow Marker ||  ||
 * Green Marker ||  ||
 * Blue Marker ||  ||

*Safety Concerns

 * 1) Wear safety goggles
 * 2) No direct contact with any solutions

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