Thin Layer Chromatography of Amino Acids

Introduction: Chromatography is a method of separating the components of a mixture based on their differential affinity for two chemicals, one of which is immobilized (the stationary phase) and the other mobile (the mobile phase). As the mobile phase travels across a layer of stationary phase, it will carry with it components of the mixture. Those components that interact well with the mobile phase but poorly with the stationary phase will travel with the mobile phase; those that interact poorly with the mobile phase but strongly with the stationary phase will not travel as quickly. As the mobile phase moves, then, it carries components of the mixture at different rates. When the mobile phase is stopped, different components will have traveled different distances. Interactions between the components of the mixture and the stationary and mobile phases may include charge interactions or interactions between polar substances, including hydrogen bonding interactions. For instance, a TLC system can be set up with a very polar stationary phase and a nonpolar solvent. When a mixture containing substances of varying polarity is added to the mobile phase and carried across the stationary phase, the more polar compounds within the mixture will not travel as quickly. The less polar substances in the mixture will, over a limited amount of time, travel farther. Thin layer chromatography uses a solid stationary phase coated onto a glass, aluminum, or plastic support. Samples are placed on one end of the coated sheet. When the edge of the sheet is placed into a beaker of solvent, the solvent travels up the TLC sheet, like a wick, picking up and carrying components of the mixture with it. Our TLC sheets are coated with silica gel, which is very polar. We will be using a mobile phase composed of ethanol and acetic acid in a 90:10 ratio. The mobile phase is more nonpolar than the stationary phase. The system will be used to separate amino acids based on their degree of polarity. TLC is not used frequently by biochemists, except to do amino acid analyses. However, more sophisticated chromatographic methods are used all the time in clinical, agricultural, forensic, biochemical and biological laboratories. The separation principles are the same in all types of chromatography: gas chromatography (GC), high performance liquid chromatography (HPLC), and column chromatography. These methods are often used not only to separate mixture components but also to identify them (based on their separation behavior under controlled circumstances): finding drugs in blood or urine samples, finding poisons, detecting flame accelerants in arson investigations, etc. Stationary phase and mobile phase interactions can be of many types, including those based on molecular size or even based on interactions between antigens and antibodies.

B. Chromatographic separation
Materials: 1% amino acid solutions: lysine, serine, and phenylalanine unknown amino acid solution(s) 0.2% ninhydrin solution in spray bottles (use in the hood) chromatography solvent: 90:10 ethanol/ 1 M acetic acid TLC plates (approximately 5 cm x 10 cm) Special equipment: 400 mL beaker filter paper (Whatman 1) spotting capillaries for solutions (melting point capillaries are too big) rulers hair dryer latex or nitrile gloves Procedure: 1. Because fingerprints can leave significant quantities of protein and therefore ruin the TLC plate (and your results), you must wear gloves for this experiment. Obtain a TLC sheet and handle by the edges to avoid touching the coated surface. Draw a light pencil line about 1.52 cm from the bottom of the plate across the narrow side. Evenly space 4 marks across the pencil line. These will be the places where you spot your amino acid samples on the plate. Label each mark so that you can identify them later. Your TLC plate should look similar to the one shown below:

Lys

Ser

Phe

unk

2. Spot a small sample of amino acid solution onto each mark with a capillary tube. Spots should be small: more is not better here! The method is very sensitive. Large spots lead to imprecise results. Repeat (you have a total of two small spots of each sample). Allow the plate to dry. 3. Add enough solvent to the bottom of a large beaker so that the entire bottom edge of the TLC plate will be sitting in solvent (about 1 cm in the bottom). The solvent in your beaker cannot be above the starting line on your TLC plate! Place a piece of filter paper along the side of the jar and wet with solvent. Prepare a piece of plastic wrap to cover the top. 4. Place the TLC plate in the beaker, sample side down. Cover with plastic wrap or a watch glass. Usually, one allows the solvent to travel up the plate until it has traveled 85% of the way to the top (within a couple of centimeters). However, this may take more than one hour. Instead, leave the TLC plate undisturbed in the beaker for 40 minutes. That is sufficient time for good separation of the amino acids in this experiment. 5. Remove the TLC plate carefully and immediately draw a thin pencil line at the solvent front: the line which defines how far the solvent has traveled. Allow the chromatogram to dry completely. 6. Take the plate to the hood, spray lightly, but evenly, with a ninhydrin solution. Be careful to keep the spray in the hood: avoid inhaling it or getting it on your hands. Allow to dry, and heat with a hair dryer. Keep your finger on a corner to prevent the hair dryer from blowing the plate away. Amino acids present in the mixture should appear as purple or orange/brown spots. Draw a circle around the outside edge of each spot. Mark the center of each spot with a pencil. 7. Your chromatogram will look similar to the one shown below. The spots wont be in the same place because we have intentionally put them in the wrong place in the example below so we dont give you the answer before you have a chance to work on it. Also, your spots may not be circular; they may be elongated or elliptical.

Lys

Ser

Phe

unk

8. Measure the distance (cm) from the starting line to the top of the solvent line to obtain the distance traveled by the solvent. 9. Measure the distance (cm) from the starting line to the center of each amino acid spot. 10. Calculate Rf values for each amino acid according to this equation:

Rf =

distance traveled by compound (cm) distance traveled by solvent (cm)

Record Rf values for each amino acid you test as well as the unknown. Identify the substance(s) present in the unknown. See the worked example below:

Solvent line

Rf calculations: Lys
7.51 cm

5.68 = 0.756 7.51 4.61 = 0.614 7.51 3.27 = 0.435 7.51 3.27 = 0.435 7.51

Ser Phe

5.68 4.61 3.27 3.27

unk
Starting line

Lys

Ser

Phe

unk

In this example, it is obvious that the unknown is simply phenylalanine. You may have a mixture of one, two, or three amino acids in your unknown.

Report Sheet for Part B

B. TLC Chromatography of Amino Acids
Chromatogram drawing (by hand or using ChemDraw)

Calculation of Rf Values Distance (cm) from the starting line to the solvent line: Amino Acid Lysine Serine Phenylalanine Distance (cm) amino acid traveled Rf value

Unknown(s)

Unknown #

Identification of unknown(s):

Use the table of amino acids at the end of this handout to help you answer Q.1 and Q.2. Q.1 Which amino acid traveled the furthest up the TLC plate? Why?

Q.2

Which amino acid traveled the least? Why?

Q.3

Why must you use a pencil to write on the TLC plate (starting line, solvent line, identification of the spots)? Why cant you use a pen?

Neutral, nonpolar side chains O H3N CH C O H Glycine (Gly) O H3N CH C O CH CH3 CH2 CH3 Isoleucine (Ile) Phenylalanine (Phe) Proline (Pro) CH2 H2N CH3 CH3 Alanine (Ala) O H3N CH C O O H3N CH C O CH CH3 Valine (Val) O C O CH2 CH2 S CH3 Methionine (Met) O H3N CH C O CH2 H3C CH CH3 Leucine (Leu) O H3N CH C O O H3N CH C O

O H3N CH C O CH2 OH Serine (Ser)

Neutral, polar side chains O O H3N CH C O CH CH3 OH Threonine (Thr) OH Tyrosine (Tyr) HN H3N CH C O CH2 CH2

O H3N CH C O

Tryptophan (Trp)

O H3N CH C O CH2 SH CH2

O H3N CH C O C O NH2 CH2 CH2

O H3N CH C O

C O NH2 Glutamine (Gln)

Cysteine (Cys)

Asparagine (Asn)

Basic, polar side chains O H3N CH C O CH2 HN NH CH2 CH2 CH2 CH2 NH3 Histidine (His) Lysine (Lys) O H3N CH C O CH2 CH2 CH2 NH C NH2 NH2 Arginine (Arg) O H3N CH C O

Acidic, polar side chains O H3N CH C O CH2 C O O CH2 CH2 C O O Aspartate (Asp) Glutamate (Glu) O H3N CH C O