Spinach Paper Chromatography

Proteins consist of chains of smaller compounds called amino acids, they contain carbon, hydrogen, oxygen, nitrogen and usually sulfur atoms. Enzymes, antibodies, and the myoglobin are examples of proteins (American Heritage: Student Science Dictionary 272). Protein is a straight chain of amino acids. You need protein to survive (Rader).

The macromolecule protein is made up of monomers called amino acids which are nitrogen-containing molecules (Hoffman and Falvo, 2004). These amino acids are joined together by peptide bonds. Amino acids can be categorized into three types called essential amino acids, semi-essential amino acids and non-essential amino acids. Essential amino acids (e.g.: valine and phenylalanine) refer to amino acids that are not synthesized in the human body; hence it has to be provided in the diet. Semi- essential amino acids represent amino acids that are synthesized in the body but not in adequate amounts; therefore it has to be supplied by the diet in small amounts. Non- essential amino acids (e.g.: alanine and glycine) are synthesized by the body and

Amino acids are mainly composed of an amine (-NH2), a carboxylic acid (-COOH), an atom of hydrogen (-H) and a

As stated on Wikipedia proteins are large biomolecules, these are any molecule that is present in living organisms such a carbohydrates , or macromolecules which are molecules that are a necessity for life which includes carbohydrates also. Proteins consist of one or more long chains of amino acid. Amino acids are acids that help break down the food we eat to make it into another type of protein, there are many different names of amino acids. The basic strain of proteins is made up of amino acids and peptides. There are 3 different types of protein structures; primary, secondary and tertiary.

You can use thin-layer chromatography for quantitative analysis to some extent. If you use solutions of known concentrations and compare the unknown to the standards, then you can gain some quantitative information. You can also calculate Rf values taking the distance traveled divided by the total distance of the eluent. After we chopped up our spinach leaf we used hexane and ethanol to separate out compounds in our leaf. After adding water, we removed the top layer and spotted a TLC plate to separate out our compounds. We also used a mixture of hexane/ethyl acetate (1:1) and hexane/ethyl acetate (20:1) to use as our developing solution. The (1:1) solution gave use six compounds separated out on a TLC plate with xanthophyll being the most polar and carotene being the least polar. On the other hand, the (20:1) solution gave us one compound that moved through our TLC plate. Looking at the plate it seems like it is the carotene that moved while everything else remained near the polar side of the plate. The most polar molecule was Rhodamine B (bright pink-red) then Fast Green (blue) then we had a clear compound show up, then Bismark Brown (yellow-orange) and the least polar compound was Sudan IV

Before the experiment was conducted, a chloroplast solution was created which was comprised of deveined spinach leaves. Deveining the leaves cuts off the supply of food and water that is carried to the leaves and the veins do not carry as much chloroplast compared to the leaves. The leaves were placed under a lamp to absorb any remaining fluids and then added to a blender that had been chilled to slow down respiration of the spinach leaves and enzymatic processes within the spinach leaves that cause browning. The solution was blended in three 10-second bursts after a .5 M sucrose solution was added to the spinach leaves.

Once the application of the spinach extract was finished, acetone, the solvent, was poured into the beaker. Then the cylinder was placed into the beaker to absorb the solvent with a jar top placed on top of it to hold it in place. The chromatography cylinder stayed in the beaker until the pigments in the spinach extract had been separated into different colors and was about 4-5 cm from the top of the paper. After this, the chromatography cylinder was removed to dry for the different pigments could be observed.<p>

The unknown substances found using both methods of chromatography could be identified using the calculated retention factor (Rf) values. The equation is below: The Rf values for the substances on the paper chromatograms were calculated using this equation as follows: The Rf values for the substances on the thin-layer chromatograms were also calculated as follows: Below is a table containing the colour and retention factors of different substances found in plants: The retention factor of a compound changes from laboratory to laboratory, and even from experiment to experiment. This is due to any slight variations in the setup, the mobile and stationary phases used, and the temperature. From this investigation, the pigments separated from the plant extract can be identified using their colours, along with the Rf values.

Proteins are macromolecules that contains nitrogen, carbon, hydrogen, and oxygen. Proteins are polymers of molecules called amino acids. They are compounds that are made up of ( -NH2) on one end and then ( -COOH ) on the other end. More than 20 different amino acids are found in nature. All amino acids are identical in the regions where they may join together by covalent bonds. Some proteins are used to control the rate of reactions and regulate cell processes. Some are used to form bones and muscles. Others transport substances into or out of cells or help to fight diseases.

The solvents that were used in the column chromatography were hexanes, 75/25 hexanes/acetone solvent, and acetone. The adsorbent used in the column chromatography was alumina, which is polar. All of the pigments moved through the column based on their polarity. Hexanes were used because they are nonpolar due to their hydrocarbon chain and was used as the initial solvent before the extract was placed in the Pasteur pipette. Carotene, which is nonpolar hydrocarbon chain, was the first pigment extracted. The nonpolar solvent was used because it is soluble in the nonpolar carotene, thus moving it through the column quickly. To elute the next pigment of chlorophyll the solvent was the hexane acetone mix. This mixture is slightly more polar because

During the first part of the experiment a dot of ink was placed onto chromatography paper to separate the mixture by pigment. This chromatography paper was place so that a portion of the paper below the dot was in water, so that the water could travel up the paper and separate the ink. The paper was suspended using a toothpick that was put through the top of the paper, which was held by the neck of the bottle.

Thin layer chromatography can be used as a physical method to segregate compounds from natural sources. E.g. Spinach leaves are visibly green, but consist of a variety of components that have more colour than others. This experimental procedure uses compounds from spinach leaves that are exposed to chromatography, TLC plate to indicate the different pigments

With a given 10x20 Whatman #1 chromatography paper, the dots were marked and numbered from 1 to 9 in pencil within 2 cm intervals from each edge, 2 cm along the lines, and 2 cm from the bottom. With a scrap piece of chromatography paper, 8 substances were obtained; Red #3 (erythrosine), Red #40 (Allura red), Blue #1 (brilliant blue FCF or erioglaucine), Yellow #5 (tartrazine), Red food coloring, blue food coloring, green food coloring, and yellow food coloring. And the last ninth substance was needed to made with brown M & Ms. The 2 brown M & Ms were left on the beaker mixed with 0.5 mL water and 0.5 mL ethanol. Once the brown color disappeared from shells of the M & Ms to the solvent, the liquid changed from clear to dark brown color.

In order to proceed with the lab, we must know what chromatography is and how it works. Chromatography is used for the separation of a dye mixture. As mentioned, there are two important components of chromatography; the adsorbent and the eluent. Adsorbents are the solid materials that attract and absorb the materials to be separated. The eluent is the solve, that carries the materials to be separated through the adsorbent. The concept of chromatography is that the compounds that are to be separated are slightly soluble in the eluent and spend some time on the adsorbent. Therefore, when the components of the mixture have different affinities for the eluent, they can be separated from each other. This works because of the polarity of the molecules

This experiment will will need several materials. Materials include: spectrophotometer, spinach leaf, chromatography paper, coin, and a test tube containing containing chromatography solvent, cuvette, and water.