Thin Layer Chromatography – Amino Acids (the monomers of proteins!)

Background

Proteins

Proteins are a diverse group of biological molecules that perform a variety of functions for living organisms. Some proteins are structural, such as those that make up human hair, animal hooves and tendons and some proteins perform highly specialized functions inside a cell based on their shape and chemical composition.

Proteins are large polymers made of monomers called amino acids. Almost all proteins in life are made of the same group of 20 amino acids as shown in Fig. 3-5 below. Think of them as being like 20 different Lego shapes. Not all of them have to be used to make a structure, but there are nearly an infinite number of possible sculptures that can be made from using only 20 lego shapes. All 20 amino acids have the same basic formula, a central carbon with an amino group, a carboxyl group, a hydrogen and only the “R” group will differ between each amino acid. The “R” groups will have a significant influence on the final shape of the protein

In today’s lab you will be using a technique to separatea group of unknown amino acids in a solution based on their size and overall charge.

A similar technique is also employed by forensic scientists to identify different proteins on a crime scene.

Thin Layer Chromatography (TLC)

Chromatography is defined in Chemistry Principles & Practice by Reger et al. as“ a technique that employs two materials, one moving and one stationary, to separate a mixture into its components.” The basic idea is to have a solvent, such as water, move through a stationary medium such as paper. For example, if you hold a paper towel in a dish of water such that only a small portion of the paper towel’s edge is submerged in the water, the waterwill move up the paper towel and saturate it.

There are many different types of chromatography techniques available to ascertain the contents of unknown substances. For example, gas chromatography was used on the Mars Rover to determine the chemical components of Mars’s surface whereby a carrier gas, a bit like the water in the example above, is pushedthrough a narrow tube. The unknown substances are then added to the gas. Different molecules will move at different speeds through the gas filled tube. These molecules will also separate in the tube based on their size and charge, causing each substance to separate from the original mixture and exit the tube at a different time. The time involved in separation can be measured and is compared with known values for known substances.

Today in lab we will use a plastic strip coated with a cellulose absorbent (a thin sheet of plastic coated in paper). Like the gas that carries substances through a tube in the example above, and the water in the paper towel example, you will use a special liquid solvent that will move up the paper, as it does so it will carry and stain (so you can see it) any amino acids in the solution you applied to one endof the paper (see diagram provided). As the solvent moves up the paper, the unknown amino acids in your solution will separate. You will then measure the distance that each amino acid travelled, and based on known amino acid travelling distances through the same solvent, you will calculate which amino acidswere in your solution based their Rf values. To calculate the Rf values you will use the equation below and the measurements shown in the diagram.

Rf = Distance of Amino Acid spot (compound spot)

Distance solvent travelled (solvent front)

The “Rf” value will be compared with the Rf value of known amino acids measured using the same solvent and paper.

YOU MUST USE GLOVES IN THIS LAB WHEN HANDLING SUBSTANCES, YOU WILL BE WORKING WITH HAZARDOUS CHEMICALS AND ANY AMINO ACIDS ON YOUR HANDS CAN CONTAMINATE THE RESULTS!

Procedures

1. Place 25 drops of the mixed developing solvent provided into the glass developing chamber and place the cap on topAvoid inhaling or getting on your hands! Use gloves! This will saturate the atmosphere of the chamber insuring better separation.

2. With a pencil (NOT a pen) very lightly mark a small dashed line approximately 1 cm up from the bottom of the cellulose paper strip provided, then place one very small spot of Amino Acid unknown #1 or Amino Acid unknown #2 on your dashed line using the capillary tube provided (teeny tiny glass straw). *USE gloves whenever you handle the cellulose strip!! Also be sure to keep your finger on the open end of the capillary tube in order to control the size of the drop.

3. Repeat spotting in the exact same location as the first spot 5 more times allowing each spot to dry completely before adding the next.

4. After completing the repeated spotting, allow the TLC strip to dry completely, then place the paper into the developing chamber and replace the cap(USE GLOVES). Try to be sure the strip is placed diagonally in the chamber so the top of the paper rests against the edge of the chamber opposite the bottom.

5. Allow the developing spot to migrate until it reaches about 1cm from the top. Remove and air dry. Place in an oven for 5 minutes to develop.

Results

The components of the amino acid mixture can be identified by their separation on the absorbent cellulose paper and their Rf values. Calculate the Rf Value for all of your amino acids by measuring the distance the solvent travelled in cm and the distance of each amino acid spot in cm and using the equation below.

Rf = Distance of Amino Acid spot

Distance solvent travelled