Separation through Ion Exchange Chromatography
Introduction
Amino acids are important molecules that serve as building blocks for all proteins that are made in every organism. Although there are millions of different proteins that can be made by all living things combined, all proteins are derived from a base of 20 amino acids that are combined together in different ways to create the diversity of life that is on Earth. Humans cannot produce all of the amino acids needed for protein synthesis, so these essential amino acids must be consumed so that the body can function and continue to produce proteins needed for survival. All amino acids share the same basic structure, which is a carboxyl group and an amino group attached to the same carbon …show more content…
The depth of the solvent is important, if it is too deep, the spots on the TLC plates will become submerged and alter the results, too shallow and the solvent will not be able to make it to the desired height on the plate. Since these solvents are generally volatile, a piece of filter paper was added to the jar that had been soaked in the solvent in order to saturate the air inside the sealed jar to prevent evaporation from the plate. The jar was left sealed for about five minutes to ensure air saturation while the TLC plates were prepared for use. A line was drawn two centimeters from the bottom of the silica plates with a pencil (ink would be carried up the plate and interfere with results) and care was used in order to ensure that the plates were not touched, which would have caused contamination from the oils on the skin which could show up as extra spots once the plate is developed. Five marks were placed on the first plate, these marks were labeled Mix, Phe, Lys, and Ala (Figure 1). On the second plate, four marks were drawn, labelled as Mix, pH3, pH6, and pH11 (Figure 2) and this plate was then placed aside for use later in the experiment. The first plate was then spotted using capillary tubes and spotting a tiny drop of each amino acid on its labelled spot, then using the mixture to also make a spot on its mark. This plate was then sealed inside the developing jar and allowed to run until the solvent reached about ¾ of the way up the plate. Once this occurred, the plate was removed from the jar, the solvent front was marked with another pencil line, then the plate was dried on a hot plate, then sprayed with ninhydrin, a chemical that degrades the amino acid into aldehydes, ammonia, and carbon dioxide, then reacts with the ammonia to produce a purple color
Introduction
Amino acids are important molecules that serve as building blocks for all proteins that are made in every organism. Although there are millions of different proteins that can be made by all living things combined, all proteins are derived from a base of 20 amino acids that are combined together in different ways to create the diversity of life that is on Earth. Humans cannot produce all of the amino acids needed for protein synthesis, so these essential amino acids must be consumed so that the body can function and continue to produce proteins needed for survival. All amino acids share the same basic structure, which is a carboxyl group and an amino group attached to the same carbon …show more content…
The depth of the solvent is important, if it is too deep, the spots on the TLC plates will become submerged and alter the results, too shallow and the solvent will not be able to make it to the desired height on the plate. Since these solvents are generally volatile, a piece of filter paper was added to the jar that had been soaked in the solvent in order to saturate the air inside the sealed jar to prevent evaporation from the plate. The jar was left sealed for about five minutes to ensure air saturation while the TLC plates were prepared for use. A line was drawn two centimeters from the bottom of the silica plates with a pencil (ink would be carried up the plate and interfere with results) and care was used in order to ensure that the plates were not touched, which would have caused contamination from the oils on the skin which could show up as extra spots once the plate is developed. Five marks were placed on the first plate, these marks were labeled Mix, Phe, Lys, and Ala (Figure 1). On the second plate, four marks were drawn, labelled as Mix, pH3, pH6, and pH11 (Figure 2) and this plate was then placed aside for use later in the experiment. The first plate was then spotted using capillary tubes and spotting a tiny drop of each amino acid on its labelled spot, then using the mixture to also make a spot on its mark. This plate was then sealed inside the developing jar and allowed to run until the solvent reached about ¾ of the way up the plate. Once this occurred, the plate was removed from the jar, the solvent front was marked with another pencil line, then the plate was dried on a hot plate, then sprayed with ninhydrin, a chemical that degrades the amino acid into aldehydes, ammonia, and carbon dioxide, then reacts with the ammonia to produce a purple color