Introduction

The separation of simple and complex mixtures into their components, as well as the isolation of the individual components, has become an important physical method of separation in all branches of chemistry. Examples are from the research laboratory preparing new chemical compounds to the quality control laboratory testing to assure compliance with federal regulations. The different types of chromatography in common use are paper, thin-layer (TLC), column, and gas-liquid (GC, VPC, GLC).

All of the chromatographic procedures involve the interaction of a mobile phase (either a gas or a liquid) and a stationary phase (either a liquid or a solid). The four basic types are categorized in the following table:
 

TYPE	MOBILE	STATIONARY	PRINCIPLE OF SEPARATION	USES
column	liquid	solid	adsorption	Preparative scale separations
Thin-layer	liquid	solid	adsorption	Qualitative analysis & small scale separations
Gas-liquid (GLC)	gas	liquid	partition	Qualitative & Qualitative Analysis
Paper	liquid	liquid	partition	Qualitative & Qualitative analysis of polar and ionic cmpds

When the stationary phase is a solid, one is dealing with the equilibrium between the adsorption of the compound on the solid surface by adhesive forces(intermolecular bonding between the substance and the molecules of the solid phase) and its solution in the liquid phase(also intermolecular bonding but between the substance and the molecules of the liquid phase). The liquid is passing over the surface of the solid phase in one direction. In column chromatography the liquid normally moves from top to bottom (making use of gravity). In thin-layer (TLC) the liquid moves against gravity by moving by capillary action between grains or particles of the solid phase.

When the stationary phase is a liquid, we are dealing with partition equilibrium between the two fluids (the stationary liquid phase and either the liquid or gas of the mobile phase). Here we are dealing with the relative strength of intermolecular bonding with the substance with each of the fluids in the liquid or gas phase. In gas-liquid chromatography (GLC) the stationary phase is a liquid (silicon oil, carbowax, etc.) that is coating an inert solid that is packed in a tube (either glass, stainless steel or copper). In paper chromatography the liquid stationary phase is generally water that is adsorbed onto the fibers of the paper. In GLC the mobile phase is a gas (generally He is used although any inert gas will do). While in paper chromatography, it is another liquid that has different physical properties and constants from those of the stationary phase, even though the liquids themselves may be mutually soluble in one another.
 
 

In paper and TLC, you determine the Rf value of a substance under certain experimental conditions. The Rf value is the decimal value of the distance that a substance has moved (from its starting point to its final point after a fixed period of time) divided by the decimal value of the distance the solvent has moved. The solvent is measured from the same starting point as that of the substance, to the solvent fronts (the furthest the solvent has moved).

 
 

(figure 1)

In GLC the measurement is of the retention time of the substance. This is the time between injection of the sample into the GLC and the time it passes the detector. Today most detectors are coupled with a strip chart recorder and one can measure the time as a function of distance the pen has traveled from injection of the sample until a deflection peak is recorded. A graphic of the GLC function is given in Figure 2.

Under the same conditions of column temperature and carrier gas (He) flow the retention time for a substance will be the same.

Equipment

GLC, 4" x 8" piece of chromatographic paper, ruler, chromatographic chamber, stapler, capillary tubes.

Chemicals
 

Student - amino acids:	leucine, proline, histidine, alanine, unknowns (paper)
G.C. unknowns:	Hexane, methanol, n-propanol (Flammable)

Spill/Disposal

amino acids: Spill/Disposal: A
G.C. unknowns: small amounts (< 5mls) Spill/Disposal: A
developing solvent: Spill/Disposal: A

IN HOODS
developing solvent (ACETIC ACID, iso-PROPANOL) + ninhydrin (already in chromatography chambers)

Procedure

Part I. Separation of Amino Acids by Paper Chromatography
1. Take a piece (4" x 8") of chromatography paper and draw a light pencil line 3 cm from the longer edge. Place an X at 3 cm, 6 cm, 9 cm, 12 cm and 15 cm. Handle the paper as little as possible. When handling it, hold it on the edges. Under the first X place the letters Leu (this is where the known amino acid, leucine, will be applied to the paper), under the second X place the letters Ala(for alanine), under the third X place the letters His(for histidine), under the fourth X place the letters Pro(for proline), under the last X place the number or single letter of the unknown you are assigned by your lab instructor. All of these marks must be done in graphite (or lead) pencil. Do not use ink. (See Figure 3)

2. You may want to practice the spotting of a solution on paper. Practice with water and a paper towel (or a piece of filter paper). Do not waste the chromatographic paper for practicing. Place a capillary tube in the water. When you remove the capillary, you will see a small amount of solution on the inside of the capillary even when you don't stopper the upper part of the tube. Now gently touch the end of the capillary with the solution to the paper allowing only a small amount of the liquid to wet the paper. What you want is a small spot of solution on the paper--about 2mm (.2 cm in diameter). If you want more material put on the paper, you should wait until the first spot has dried before applying a second spot of the same material. When you feel you are ready, place a small spot of the leucine solution on the X that you have labeled Leu. Do the same for the remaining X's. Put a spot of the solution for which the X is labeled. Do not use the same capillary for each--you will contaminate each spot with the previous substance. Use a different capillary for each solution (or use the capillary that was present in the solution).

3. Allow the spots to dry. Carefully put the paper into the chromatography chambers that will be located in the hoods. The starting line should not be below the level of the solvent in the chromatography chamber. The developing solution can be hazardous so do not remove chromatography chambers from the hoods. The developing solution will already be in the chamber. Allow the chromatogram to develop at least one hour. The amino acids will move up the paper by capillary action at different rates depending on their relative solubilities in the developing solvent and the water on the cellulose of the paper.

4. After the solvent has risen a sufficient amount, remove the paper and mark with a pencil line to the farthest position on the paper reached by the solvent. Immediately take the paper to the drying chambers also located in a separate hood. Gently dry the paper with the heat guns provided.

5. Heat the developed paper for two to three minutes with the heat guns. The amino acids react with the ninhydrin (in the developing solution) spots. Once the spots are developed sufficiently, remove the chromatography paper from the drying chamber. Circle the spots and measure the distance each has traveled compared to the distance moved by the solvent. Record this on the data sheet. These are the Rf values. Also note the color of the spots.

6. From the Rf values of the known amino acid solutions and the Rf values of the spots of your unknown, what amino acids were present in the unknown?

Part II. Qualitative Analysis Using the A Column

1. You will be doing a gas chromatograph of a sample of a mixture that is prepared from the following compounds: hexane, methanol and n-propanol. Your instructor will run samples of the individual pure compounds.

2. The instrument conditions are:
Temperature: 115° C
Carrier gas flow: about 60 ml/min at column exit
Detector Current: 200 mA (He carrier gas)

It is important that you do not change the current, gas flow, or temperature during the experiment. Not only will it effect your results but it could cause serious damage to the instrument. Also do not touch the metal plate, where you inject your sample. Both plates--the A and B column--will be only a few degrees below the temperature of the columns, which is 110° C, so the plates are about 100-105°C. This is hot enough to blister the skin.

3. Rinse the syringe twice with the sample to be tested. (Squirt the waste rinse into the flask provided at the GC.) Draw a third syringe of the mixture sample from the sample container (2 m L or .002 mL of sample). With the recorder running at 1"/min, inject the sample into the A column. Push the needle in all the way through the rubber septum. A metal stop on the syringe will prevent the needle from going in too far. With the needle in the column depress the plunger to inject the sample. Simultaneously, you will need to push the Start button on the recorder. The instructor will set the time on the run so that the longest retained material will have cleared the instruments. Do not do this yourself, because the time varies with the temperature.
 

1. You will note that the different substances take different lengths of time to exit the instrument (reach the detector).

Your unknown graph is then compared to the known samples and you should be able to determine what is in your unknown. Record the data. Determine the amount (in mLs) of the compound that has the highest percent in your unknown.