Chromatography, a term that literally means "colored writing or picture", is a technique ordinarily used to separate a series of solutes mixed within a common solvent. There are several different types of chromatography including paper and thin layer chromatography (TLC). Chromatography is a qualitative type of procedure which tells you what type of molecule is present but not necessarily how much of each molecule is present.
Introduced in 1944, paper chromatography is a technique that involves separation and identification of chemical substances by a solvent that is moving up the sheets or strips of filter paper. A drop or spot of sample mixture (usually a solution) is placed or spotted near one end of a piece of filter paper. The drop is allowed to dry, thus leaving a spot of the mixed substances. The end of the paper nearest the spot is allowed to be in contact with a suitable solvent, WITHOUT immersing the spot itself. If the spots were immersed they would dissolve in the solvent and be pulled down into the solvent itself. In ASCENDING CHROMATOGRAPHY (the type we will be using), the solvent is in a pool at the bottom of a chamber or jar in which the paper is supported. The solvent rises up the paper by CAPILLARITY.
The solvent flows through the paper, over and past the mixture of substances. As it flows, the solutes dissolve within the moving solvent and are carried along the moving solvent. EACH SUBSTANCE WILL MOVE AT ITS OWN RATE DEPENDENT ON FACTORS SUCH AS MOLECULAR SIZE, WEIGHT, AND CHARGE.
The solvent flow is allowed to continue for a some period of time, usually until the solvent front is a predetermined distance from the top of the strip. The paper is allowed to dry and the separated substances are observed immediately, if colored, or located as spots by means of an appropriate chemical reaction. This technique is known as ONE-WAY or ONE-DIMENSIONAL CHROMATOGRAPHY and the finished paper is called a ONE-WAY CHROMATOGRAM.
FORCES AT WORK
Two types of forces are at work in chromatography. One series of forces helps to speed up the reactions or propel them. A second series works in opposition to slow down or retard them. The distance actually travelled by molecules from their starting point is the result of these two opposing forces.
Quantitative measures are used to designate the approximate position a compound occupies on a chromatogram. The term to describe this position is the Rf (Rate of Flow) value. This is the ratio of the distance travelled by the dissolved substances to the distance travelled by the solvent. Visit this site for an explanation of how chromatography works.
|Distance travelled by the solute|
|Rate of flow =||---------------------------------|
|Distance travelled by the solvent|
The Rf value is usually constant for any given set of conditions and gives a characteristic pattern that one can readily recognize or read. It should be emphasized that the Rf will vary according to the solvent used, so the solvent should always be stated when recording the Rf. Also, other factors are involved. The temperature and the nature (quality) of the paper must be considered and noted. It is possible for two different substances to exhibit the same Rf value under closely controlled conditions, so this technique is not an ABSOLUTE method of identification.
THE PROPELLING FORCES
There are two basic factors involved in the speeding up or propelling forces. One is the SOLVENT FLOW through the material that is being used. Solvent flow is the same for all the substances in a chromatogram. The second factor which has a differential effect on the reaction is SOLUBILITY. Certain molecules are more soluble in a certain solvent than others. Solvents are chosen to create the greatest possible differential in the solubilities of the substances to be separated. As no one solvent may produce maximum solubility differentials for all the substances involved, we sometimes use two solvents with different properties. These are run successively at right angles to each other. This is known as TWO-WAY or TWO DIMENSIONAL CHROMATOGRAPHY and the finished paper is called a TWO WAY CHROMATOGRAM.
THE RETARDING FORCES
There are also two major retarding forces: ADSORPTION and PARTITION. ADSORPTION refers to the ability of materials or molecules to stick to surfaces. In the case of chromatography certain molecules will stick to the cellulose of the filter paper more strongly than others. Adsorption is another of the differential forces. The release of substances from the spot will vary from substance to substance. As the chromatogram runs, the more strongly adsorbed substances are held back while the less strongly adsorbed substances move on ahead of them. PARTITION represents the degree to which the solutes divide
themselves up betwen the solvent and the fluid (usally water) which is bound into the filter paper.
- Chromatography chamber (large glass jar); can also be a glass, a wide mouth test tube or a section of a 16 or 24 oz. plastic water bottle
- Glass Petri dish bottom; can also be the bottom section of a plastic water bottle cut off about an inch from the bottom
- Whatman #1 Chromatography paper; for home coffee filters will do nicely (#4 is usually good size and quality)
- Paper towels, pencil, ruler, scotch tape, tooth picks (flat end)
- Glass jar with top (pasta sauce jars will work)
- Stapler, Heat gun, Various inks and dyes; felt ink pens and vegetable dyes will also do
- 70% or 90% Isopropyl Alcohol can be used for the inks and for the chlorophyll separations (available at local drugstore or pharmacy)
Here's another step-by-step procedure for paper chromatography.
- Here is a simple do at home version that can be done by students of any age. Another way to do the lab is to fill the Petri dish (bottle bottom) about 3/4 full with distilled water* and set carefully in the bottom of the jar. Put the top on the jar and allow the water vapor to saturate the air in the chamber (jar). *You can get distilled water at the local grocery store or take some bottled water and put it in the microwave for 45-60 seconds (until the water boils). Be sure the top was loosened first. Allow the water to cool to room temperature before you use it.
- Obtain a sheet of the special Whatman #1 Chromatography paper or use the coffee filters after you have cut them to size. Avoid touching the paper with your fingers anymore than is absolutely necessary. Touch only the sides or the top edges of the paper or hold it between a paper towel.
- Spread a clean paper towel on the table top and lay the chromatography paper on it. With a pencil and clean ruler, draw a line across the bottom of the paper, approximately 1 inch from the bottom. Make a series of small x's on this starting line. The x's should be at least one inch apart and there should be at least one x for each type of ink or dye provided for the experiment. See a variation of this technique using a radial type of spotting.
If using a strip of filter paper then draw a line or x about 1 cm from the bottom of the strip and attach the strip to a pencil or rod suspended across the top of the jar. If using chlorophyll extracts, use the IPA as the solvent.
- With the ink and dyes provided, make a small spot on the x. Here's a variation for those of us with a sweet tooth. Allow these spots to dry if necessary. Using two or three staples, curl the paper into a cylinder shape and staple it secure. You may also suspend the filter paper from a pencil sitting on the mouth of the jar (chamber) as shown in the pictures at the websites. Being very careful to avoid splashing the water in the Petri dish onto the paper, lower the paper cylinder into the water and close the chamber OR suspend the filter paper from the pencil into the water. Seal the lid of the chamber with masking tape. If using the jar and suspending the filter paper then leave the top off the jar.
- Allow the water to move up the paper until it is about an inch (2.5 cm) from the top of the paper. Then remove the paper cylinder or the suspended filter paper and with a pencil, mark the position of the solvent front. Also circle each of the areas where the pigment has separated. Dry the paper with the heat gun (hair dryer) if necessary.
If plant pigments are used instead of dyes, substitute rubbing alcohol(IPA) (highest concentration you can get) and rub the pigment from the plant onto the filter paper by rubbing a coin over the invading organism. In this case, results should look somewhat like this.
- Compute Rf values for all the spots that are identifiable on your chromatogram. To do this, measure the distance from the starting line to the top of each point and divide this number by the distance from the starting line to the solvent front. It should be obvious that in all cases, Rf < or = 1.
Turn in a copy of your chromatogram, showing pigment areas identified and corresponding Rf values (show all calculations).
Answer the following questions:
1. While chromatography refers to the presence of color, many molecules that are subjected to separation through this tecnique are colorless. Amino acids in solution can be separated through paper chromatography but are colorless. Specifically, how might we expose the amino acids in a paper chromatogram?
2. Devise an experiment, using paper chromatography to determine if a group of plants were related to each other. What procedures would you use and what results would you be looking for? (Be specific)